Zu Hauptinhalt springen
Startseite UR

Research


Fakultät für Biologie und Vorklinische Medizin
Institute of Plant Sciences
Cell Biology and Plant Biochemistry
PD Dr. Stefanie Sprunck - Research

 

In summary, our research group works on proteins contributing to cell polarity, on the gene regulatory network involved in egg cell specification, and on proteins acting on the cell surface during male-female gamete interactions.

To address questions related to our research topics we establish protocols to isolate living gametes from different flowering plant species. We perform transcriptomics and proteomics to identify candidate genes and proteins that are subsequently tested in functional studies.

Currently, we are investigating the molecular function of a small protein family that participates in the establishment and maintenance of cell polarity by characterizing specific interaction partners. Furthermore, we study RNAs and proteins putatively involved in egg cell specification, and characterize cell surface proteins which are involved in gamete adhesion, gamete activation, or fusion.

 


Transcriptomics of manually isolated gametes and zygotes

 

Transcriptomics, also referred to as expression profiling, is the investigation of a set (or all) RNA molecules expressed in a certain tissue or cell type, in a specific developmental stage, or under certain physiological conditions. Besides expression profiling, transcriptomics serves as a screening tool for candidate gene discovery.

We are manually isolating living cells from flowering plant female and male gametophytes by micromanipulation with the aim to study the transcriptome of these cells. Previously, we used female gametes (egg cells and central cells) and two-celled proembryos of wheat and Arabidopsis to perform either single-run partial sequencing of randomly selected cDNA clones (ESTs; “expressed sequence tags”) of generated cDNA libraries (Sprunck et al., 2005), or microarray-based expression profiling (Englhart et al., 2017). Meanwhile, we perform next-generation sequencing (NGS) to investigate the RNA and small RNA content of female and male gametophytic cells from different flowering plant species.

We are analyzing our transcriptome data sets with the aim to discover uncharacterized gamete-specific or early fertilization-induced mRNAs, as well as small and long noncoding RNAs. We identified, for example, transcripts specifically present in the egg cell and/or the central cell and transcripts specifically induced in zygotes and two-celled proembryos (Chen et al., 2017; Leljac-Levanic et al., 2013; Sprunck et al., 2012; Juranic et al., 2012; Sprunck et al., 2005). Currently, we investigate the molecular function of selected candidate genes/gene families participating in the establishment of cell polarity, in gamete specification, and in gamete interactions during double fertilization.

 

Bild 1


Establishment of cell polarity

 

Establishing and maintaining proper cell polarity are key aspects of life. One of the major goals of cell biology is to understand the molecular mechanisms mediating cell polarization. Generally, cell polarity means cellular asymmetry.

 

 

Polarization and asymmetrical growth in eukaryotic cells require elaborate regulation of a variety of cellular processes, including spatially restricted signaling, reorganization of the cytoskeleton, and polarized membrane trafficking.

Polarized cells divide asymmetrically, generating daughter cells that are different in cellular contents, cell size, or developmental potential.
The asymmetric distribution of cell fate determinants (e.g., proteins and RNAs) but also asymmetric translational repression and asymmetric protein degradation govern cell fate determination and differentiation, which is essential for the development of a multicellular organism or tissue, containing cells of different molecular fate.

To study the establishment and maintenance of cell polarity in flowering plants we use the following Arabidopsis model cells: pollen tubes, root hairs, trichomes, egg cells/zygotes.


1. Polar tip growth of pollen tubes

The pollen tube is among the fastest growing plant cells. It is widely used as a model cell to study tip growth.

The pollen grain germinates on the surface of the stigma and forms an elongating tube that navigates through the female tissues toward the ovule to deliver its two sperm cells for double fertilization. Like growth cones of axons, pollen tubes require a signaling network to recognize and interpret extracellular cues that determine the site and the direction of tip growth.

 

 

Within the past years, crucial molecular players of the pollen tube tip growth machinery have been identified, including a tip-focused calcium gradient, elaborate F-actin dynamics, tip-directed vesicle trafficking, and localized exocytosis. In our lab, we identified the Armadillo repeat protein ARMADILLO REPEAT ONLY 1 (ARO1) as a novel player of the pollen tube tip growth machinery (Gebert et al., 2008). Currently, we investigate the mechanistic function of ARO1 and related ARO protein family members in cell polarization.

 

2. Establishment of polarity in the egg cell


The egg cell and the zygote

The mature egg cell of the model plant Arabidopsis thaliana is a highly-polarized cell. The large vacuole is located at the micropylar pole of the egg cell, adjacent to the micropyle of the ovule (the entrance point of the pollen tube), while the nucleus and most of the cytoplasm is located at the opposing chalazal pole.

 

 

Immediately after fertilization, the vacuolar organization changes and the zygote nucleus is moved away from the chalazal pole. Live-cell imaging during the entire process of zygote polarization revealed that the preexisting alignment of microtubules and F-actin in the egg cell is lost on fertilization and that the rapidly elongating zygote reorients the cytoskeletons to perform directional cell elongation and polar nuclear migration (Kimata et al. 2016, PNAS 113, 14157-14162).

The first zygotic cell division is transverse and asymmetric, producing a smaller apical cell and a larger basal cell. The two daughter cells do not only differ in terms of their morphology but also in their developmental fate. The apical cell gives rise to the later embryo, while the basal cell mainly forms the extra-embryonic suspensor.

 

 

 3. ARO proteins and polarity

In order to identify novel players participating in the establishment of cell polarity in egg cells and pollen tubes, we performed transcriptomics approaches using isolated egg cells of wheat and Arabidopsis. We focused on uncharacterized (novel) transcripts present both in egg cell and in pollen tubes.
One of the first candidates chosen for functional analyses was a transcript encoding the Armadillo repeat protein ARMADILLO REPEAT ONLY 1 (TaARO1) and its putative ortholog in Arabidopsis (AtARO1).

ARO1 genes are specifically expressed in the pollen vegetative cell and the egg cell. In the Arabidopsis pollen tube, the AtARO1-GFP (green fluorescent protein) fusion protein localizes to vesicles, accumulating in the inverted cone-shaped region of the pollen tube tip (Vogler et al., 2015). Moreover, the accumulation of AtARO1-GFP in the pollen tube tip is sensitive to brefeldin A (BFA), a fungal metabolite that inhibits protein secretion. These observations support our hypothesis that the observed partial co-localization of AtARO1-GFP with F-actin in the shank of pollen tubes reflects the vesicle transport along longitudinal F-actin cables (Gebert et al., 2008).

 

 

We found AtARO1 to be of fundamental importance for polar pollen tube tip growth. Compared with wild type pollen tubes, the pollen tubes of AtARO1 knockout plants (aro1-3) revealed a highly-disorganized F-actin cytoskeleton and growth depolarization (Gebert et al., 2008).