Physiological studies on egg cell activation

4.2.4.1 Subcellular localisation of EC1 in Nicotiana benthamiana leaf epidermis cells

In A. thaliana wild type plants, EC1 is exclusively expressed in egg cells and localised to vesicle-like structures. To further determine the shape and localisation of these structures, the EC1.1-GFP fusion protein was transiently expressed in leaf epidermis cells of Nicotiana benthamiana under the ubiquitously active Cauliflower Mosaic Virus 35S (35S) promoter (35Sp:EC1.1-GFP). EC1.1-GFP was localised in vesicle-like structures, further referred to as EC1-labelled bodies, in the cytoplasm. In a previous study it was shown that these EC1-labelled bodies neither co-localised with fluorescent markers for peroxisomes nor with the endoplasmic reticulum (ER; Kraus 2015). However, indications for a co-occurrence of these bodies with the ER were given and co-expression studies with markers of Golgi bodies (cis-Golgi) revealed partial co-localisation. Figure 4.11 shows confocal images (imaged by Michael Kraus) of tobacco leaf epidermis cells with fluorescent cis-Golgi bodies marked in magenta (magenta arrow; G-rb, CD3-968; Nelson  et  al.,  2007) co-expressing with EC1.1-GFP marked in cyan. The CLSM images were analysed in more detail and revealed that EC1-labelled bodies appear not only as single dots (cyan arrow in A), but are in most of the cases

0 sec

5 sec

10 sec

15 sec

20 sec

ar ea in µm 2 5 4 3 2 1 0 EC1-labelled bodies bodiesGolgi

1 2

A

B

C

D

E

F

The images show single confocal planes of tobacco leaf epidermis cells co-infiltrated with EC1.1- GFP (35Sp:EC1.1-GFP; cyan) and cis-Golgi localised mCherry (G-rb CD3-968; magenta). (A) Overview of an epidermis cell. (B-D) Detailed images with EC1-labelled bodies (cyan arrow) and

cis-Golgi network (magenta arrow). Associated structures of EC1.1-GFP and cis-Golgi (EC1-

positive Golgi bodies) are marked with a white arrow. (E) Time lapse of moving EC1-labelled bodies are marked with cyan lines and the cis-Golgi network is marked with magenta lines. Co- migration of associated EC1.1-GFP and cis-Golgi structures are marked with white lines. The rectangle markes the separation of a previously co-migrating EC1.1-GFP and cis-Golgi. The circle marks simultaneous dissociation (1) and association (2) of two cis-Golgi networks from a EC1-labelled body. (F) The sizes of EC1-labelled bodies and cis-Golgi networks were measured with ImageJ. The median particle size of EC1-labelled bodies is 0.26 µm2 _{and the median particle} size of the fluorescent cis-Golgi network is 0.66 µm2_{. The confocal images were made by Michael} Kraus. Scale bars in (A and E) 10 µm and in (B to D) 1 µm.

R

ESUL

TS

associated to, but not overlapping with, the cis-Golgi (white arrows). These composite structures were referred to as EC1-positive Golgi-bodies.

In time lapse images (Figure 4.11, E), the parallel movement of EC1.1-GFP-labelled bodies and Golgi-bodies could be followed (white lines). Manual tracking of EC1-positive Golgi-bodies with ImageJ revealed a velocity of about 0.15 µm/sec (SEM=0.062; n=15). Furthermore, the image 4.11E also shows the separation of an EC1-positive Golgi-body between the time points 5 and 10 seconds (rectangle), where an EC1.1-GFP-labelled body moved into a different direction (cyan line) than the cis-Golgi (magenta line). Additionally, an EC1.1-GFP-labelled body, associated to one cis-Golgi body, was transferred to become associated to another cis-Golgi. Between the time points 5 and 15 seconds (circle), a cis-Golgi body (Number 1) dissociated from, while another cis-Golgi body (number 2) associated with the EC1.1-GFP-labelled body.

For particle analysis with ImageJ, fluorescence images of both fluorescence channels (GFP and mCherry) were processed separately to binary images using the RenyiEntropy method followed by a watershed algorithm to separate touching particles. In the analysis, it was not distinguished between single or associated EC1.1-GFP-labelled bodies and cis-Golgi bodies. EC1.1-GFP-labelled bodies with a median area of 0.26 µm2 _{were comparably smaller than cis-Golgi bodies with a median}

area of 0.66 µm2 _{(Figure 4.11). As EC1-labelled bodies showed no direct overlay with the cis-Golgi,}

it is hypothesized that EC1.1-GFP proteins accumulate at the trans-face of the Golgi stack or at the

trans-Golgi network, the organelle responsible for sorting secretory pathway proteins to their final

destinations.

4.2.4.2 Calcium ionophores cannot stimulate EC1 secretion in Arabidopsis thaliana

In A. thaliana, EC1 is secreted during double fertilisation (Sprunck et al., 2012), but the trigger for this secretion is not known yet. As intracellular Ca2+ _{spiking was found to play an important}

role during double fertilisation, it was suggested that Ca2+ _{may be involved in egg cell activation}

(Denninger et al., 2014). Therefore, several different Ca2+ _{ionophores were tested upon their effect}

on egg cell activation.

Ovules from homozygous plants expressing EC1.1p:EC1.1-GFP were used to monitor EC1-GFP secretion from egg cells. In mature egg cells, the fusion protein EC1.1-GFP is localised to vesicle-like structures in the cytoplasm (Figure 4.12, A and B, arrows). As shown in Figure 4.12, C and D, the fluorescence of the fusion protein EC1.1-GFP was visible extracellularly (arrowheads) after gamete fusion. The sperm cell nuclei (asterisks), marked with HTR10-mRFP, were incorporated by the egg cell. The activated egg cells expressing EC1.1-GFP can be recognized by extracellular GFP signal. Therefore, ovules were dissected and incubated in 100 mM phosphate buffer pH 7.5 containing 1% DMSO as a control and additional 100 µM ionomycin calcium salt, 100 µM A23187

or 100 µM A23187 hemi-calcium salt for 30-60 min before imaging (Figure 4.12, E to H). The egg cells showed neither a secretion of EC1.1-GFP nor obvious differences in protein localisation (n=41), suggesting that the application of calcium ionophores does not trigger EC1 secretion. In summary, egg cell activation of A. thaliana could not be stimulated by calcium ionophores, while the sperm cells were activated by application of cGMP derivates. Furthermore, the synthetic EC1.1 peptide S2 induced endocytosis in TET9-GFP labelled sperm cell plasma membranes. Moreover, ectopic expression of EC1.1-GFP in tobacco leaf epidermis cells suggests that the fusion protein mainly accumulates at the trans-Golgi network in these cells.

*

*

*

*

dsyn

ec

ec

n

vac

A

B

E

F

G

H

C

D

Figure 4.12 EC1.1-GFP protein localization in ionophore treated ovules.

The images show single confocal planes of ovules from EC1.1p:EC1.1-GFP-expressing plants. The fluorescence of the fusion protein EC1.1-GFP in egg cells is depicted in green. The preprarations were made in 100 mM sodium phosphate buffer pH 7.5. (A and B) EC1.1-GFP is expressed in egg cells and localised in vesicle like structures (arrows). (C and D) Pistils were hand-pollinated with

HTR10p:HTR10-mRFP-expressing pollen. 6-8 hap sperm cells, sperm cell nuclei depicted in red, fuse with the egg cells. The EC1.1-

GFP fusion protein is more evenly distributed in the egg cell and additionally localised outside the egg cell. (E-H) Ovules incubated in 1% DMSO (E), 1% DMSO + 100 µM ionomycin calcium salt (F), 1% DMSO + 100 µM A23187 (G), 1% DMSO + 100 µM A23187 hemicalcium salt (H) for 30 -60 min. The EC1.1-GFP derived signal was only detected inside the egg cells. Abbreviations: n, egg cell nucleus; vac, egg cell vacuole; ec, egg cell; dsyn, degenerated synergid cell. Scale bars 5 µm.

R

ESUL

TS