Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in <em>Arabidopsis</em> Double Fertilization

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...

chiba university

Angiosperms have a unique sexual reproduction system called "double fertilization." One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis.

In the double fertilization of angiosperms, one sperm cell fertilizes an egg cell to produce a zygote, whereas the other sperm cell fertilizes a central cell to give rise to an endosperm. There is little information on gamete membrane dynamics during double fertilization even though the cell surface structure is critical for male and female gamete interactions. In a recent study, we analyzed gamete membrane behavior during double fertilization by live-cell imaging with Arabidopsis gamete membrane marker lines. We observed that the sperm membrane signals occasionally remained at the boundary of the female gametes after gamete fusion. In addition, sperm membrane signals entering the fertilized female gametes were detected. These findings suggested that plasma membrane fusion between male and female gametes occurred with the sperm internal membrane components entering the female gametes, and this was followed by plasmogamy. 

Gamete membrane dynamics during double fertilization in Arabidopsis.

Fertilization requires recognition, attachment, and membrane fusion between gametes. In metazoans, rapidly evolving surface proteins contribute to gamete recognition and adhesion. Flowering plants evolved a double fertilization process wherein two immotile sperm cells are delivered to female gametes by the pollen tube, guided by elaborate communications between male and female reproductive organs. Once released, the sperm cells contact female gametes directly prior to gamete fusion. It remains unclear whether active gamete recognition and attachment mechanisms are required for double fertilization. Here, we provide functional characterization of Arabidopsis GAMETE EXPRESSED 2 (GEX2), which encodes a sperm-expressed protein of unknown function. GEX2 is localized to the sperm membrane and contains extracellular immunoglobulin-like domains, similar to gamete interaction factors in algae and mammals. Using a new in vivo assay, we demonstrate that GEX2 is required for gamete attachment, in the absence of which double fertilization is compromised. Ka/Ks analyses indicate relatively rapid evolution of GEX2, like other proteins involved in male and female interactions. We conclude that surface proteins involved in gamete attachment and recognition exist in plants with immotile gametes, similar to algae and metazoans. This conservation broadens the repertoire of research for plant reproduction factors to mechanisms demonstrated in animals.

Gamete attachment requires GEX2 for successful fertilization in Arabidopsis.

Sex-possessing organisms perform sexual reproduction, in which gametes from different sexes fuse to produce offspring. In most eukaryotes, one or both sex gametes are motile, and gametes actively approach each other to fuse. However, in flowering plants, the gametes of both sexes lack motility. Two sperm cells (male gametes) that are contained in a pollen grain are recessively delivered via pollen tube elongation. After the pollen tube bursts, sperm cells are released toward the egg and central cells (female gametes) within an ovule ( Fig. 1 ). The precise mechanism of sperm cell movement after the pollen tube bursts remains unknown. Ultimately, one sperm cell fuses with the egg cell and the other one fuses with the central cell, producing an embryo and an endosperm, respectively. Fertilization in which 2 sets of gamete fusion events occur, called double fertilization, has been known for over 100 y. The fact that each morphologically identical sperm cell precisely recognizes its fusion partner strongly suggests that an accurate gamete interaction system(s) exists in flowering plants. 

Gamete attachment process revealed in flowering plant fertilization.

Cassava ranks fifth among the starch producing crops of the world, its annual bioethanol yield is higher than for any other crop. Cassava cultivar KU50, the most widely grown cultivar for non-food purposes is susceptible to Sri Lankan cassava mosaic virus (SLCMV). The objective of this work was to engineer resistance to SLCMV by RNA interference (RNAi) in order to increase biomass yield, an important aspect for bioethanol production. Here, we produced transgenic KU50 lines expressing dsRNA homologous to the region between the AV2 and AV1 of DNA A of SLCMV. High level expression of dsRNA of SLCMV did not induce any growth abnormality in the transgenic plants. Transgenic lines displayed high levels of resistance to SLCMV compared to the wild-type plants and no virus load could be detected in uninoculated new leaves of the infected resistant lines after PCR amplification and RT-PCR analysis. The agronomic performance of the transgenic lines was unimpaired after inoculation with the virus as the plants presented similar growth when compared to the mock inoculated control plants and revealed no apparent reduction in the amount and weight of tubers produced. We show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgene specific siRNA. The results demonstrate that transgenic lines exhibited high levels of resistance to SLCMV. This resistance coupled with the desirable yield components in the transgenic lines makes them better candidates for exploitation in the production of biomass as well as bioethanol. 

Resistance to Sri Lankan cassava mosaic virus (SLCMV) in genetically engineered cassava cv. KU50 through RNA silencing.

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.

Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism.

Gamete fusion is a core process of sexual reproduction and, in both plants and animals, different sex gametes fuse within species. Although most of the molecular factors involved in gamete interaction are still unknown in various sex-possessing eukaryotes, reports of such factors in algae and land plants have been increasing in the past decade. In particular, knowledge of gamete interaction in flowering plants and green algae has increased since the identification of the conserved gamete fusion factor generative cell specific 1/hapless 2 (GCS1/HAP2). GCS1 was first identified as a pollen generative cell-specific transmembrane protein in the lily (Lilium longiflorum), and was then shown to function not only in flowering plant gamete fusion but also in various eukaryotes, including unicellular protists and metazoans. In addition, although initially restricted to Chlamydomonas, knowledge of gamete attachment in flowering plants was also acquired. This review focuses on recent progress in the study of gamete interaction in volvocine green algae and flowering plants and discusses conserved mechanisms of gamete recognition, attachment, and fusion leading to zygote formation.

Gamete Dialogs in Green Lineages.

KEY MESSAGE : hap2-1 pollen tube ovule targeting. Upon pollination, a pollen grain germinates to produce a pollen tube, which grows through the style to deliver two immobile sperm cells to the female gametophyte. Double fertilization is completed after the pollen tube enters an ovule. GENERATIVE CELL SPECIFIC 1 (GCS1)/HAPLESS 2 (HAP2) contributes to the fusion of gametes at fertilization and has been suggested to affect pollen tube guidance. However, there is controversy over the role of GCS1/HAP2 in pollen tube guidance because of conflicting results from different studies. To characterize the effects of the gcs1/hap2 mutation on pollen tube behavior, we analyzed the Arabidopsis thaliana hap2-1/HAP2 mutant, which carries a gcs1/hap2 mutation in the quartet background. The quartet mutant produces tetrads consisting of four pollen grains that remain adherent after the pollen mother cell has completed meiosis. Thus, a hap2-1/HAP2 tetrad contains hap2-1 and HAP2 pollen grains in a 2:2 ratio. Moreover, the hap2-1 locus is linked to the β-glucuronidase (GUS) gene. An excess pollination experiment with hap2-1/HAP2 tetrads revealed that the hap2-1 pollen tube targets ovules normally. Additionally, the results of restricted pollination and aniline blue staining indicated that there are no significant differences between the ovule-targeting frequencies of pollen tubes from hap2-1/HAP2 and HAP2/HAP2 tetrads.

Loss of GCS1/HAP2 does not affect the ovule-targeting behavior of pollen tubes.

All flowering plants exhibit a unique type of sexual reproduction called 'double fertilization' in which each pollen tube-delivered sperm cell fuses with an egg and a central cell. Proteins that localize to the plasma membrane of gametes regulate one-to-one gamete pairing and fusion between male and female gametes for successful double fertilization. Here, we have identified a membrane protein from  generative cells using proteomic analysis and have found that the protein is an ortholog of  DUF679 DOMAIN MEMBRANE PROTEIN 9 (DMP9)/DUO1-ACTIVATED UNKNOWN 2 (DAU2). The flowering plant DMP9 proteins analyzed in this study were predicted to have four transmembrane domains and be specifically expressed in both generative and sperm cells. Knockdown of  resulted in aborted seeds due to single fertilization of the central cell. Detailed imaging of -knockdown sperm cells during  and semi- double fertilization revealed that DMP9 is involved in gamete interaction that leads to correct double fertilization.

The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants.

Angiosperms possess a double fertilization system for sexual reproduction. Double fertilization is regulated by interactions among proteins localized in the plasma membrane of each sex gamete. A few plasma membrane resident proteins regulating double fertilization have been identified in male gametes. In contrast, no fertilization regulators in female gamete plasma membrane have been identified, largely due to difficulties in the isolation and collection of female gametes. We had produced Arabidopsis transgenic plant pDD45::GFP-AtPIP2;1 where the egg cell plasma membrane was specifically labeled with GFP (Igawa et al. 2013). The protein extract derived from approximately 200 pistils, which contained unfertilized and mature egg cells, was subjected to immunoprecipitation using anti-GFP antibody. As a result, both GFP and AtPIP2;1 were specifically detected in immunoprecipitated proteins from pistil tissues of pDD45::GFP-AtPIP2;1 transgenic plant, but not in those of wild type pistils. It was revealed that specific proteins expressed in the egg cells were successfully isolated from pistil cell population. The method described here showed the feasibility of isolating specific egg cell plasma membrane protein without gamete isolation and collection procedures.

Tomoko Igawa

Graduate School of Horticulture

chiba university

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Tomoko Igawa

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Graduate School of Horticulture

chiba university

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Graduate School of Horticulture