Embedded Files
Arabidospsis ovules are found in two chambers within the pistil, one on either side of the central septum. The ovules are connected to the septum by a stalk called a funiculus. The orientation of each ovule relative to the axis of the pistil is random
The images on this page are all from developing Arabidopsis pistils that were high-pressure frozen and then embedded within a hard plastic resin. 1 𝜇m thick sections were stained with toluidine blue then imaged on a light microscope.
This image and the next are both slices through mature ovules, but the orientation of the ovule is rotated by ~90° relative to each other. The funiculus (F) is attached to the ovule at the chalaza (C), where all the pieces of the ovule meet. The outer (O) and inner (I) integuments each are comprised of 2 layers of cells, and they grow up around the nucellus (N) and meet at the micropylar end (M) - this is where the pollen tube will enter to fertilize the egg.
The nucellus surrounds the embryo sac - the developing gametophyte - and is highly asymmetrical: on one side (the left side in this image and out towards the viewer in the previous image) it is thick, and the cells overlap like scales. On the other side it is thin, and the embryo sac is pressed up against the integuments without any empty space
The female gametophyte - called the embryo sac - develops inside the ovule. While the ovule is diploid maternal tissue, the embryo sac is haploid, and generates the egg that will be fertilized. As shown in the image above, it develops first as a syncytium, the cellularized - that is to say, first the nuclei (black dots) divide, and migrate to opposite ends of the cell, separated by a vacuole that forms (gray oval). Then cell walls form between the nuclei, creaing the individual cells, one of which is the egg cell. Two nuclei are left in the syncytium of the central cell, and they fuse to form the secondary endosperm nucleus. The developmental stage of the embryo sac can be determined based on the number and position of the nuclei, and is divided into stages FG1 through FG7, following the description by Christensen, C.A., King, E.J., Jordan, J.R., and Drews, G.N. (1997)
"Megagametogenesis in Arabidopsis wild type and the Gf mutant." Sex. Plant Reprod. 10: 49–64
Megaspore Mother Cell
The embryo sac develops from a single hapliod cell called the megaspore. The megaspore is generated by meoisis from the diploid megaspore mother cell. This image is of a very young ovule with a megaspore mother cell prior to meiosis. The funiculus is at the lower left. Just above it on both sides are the two integument primordia, and above those is the nucellus, which holds the large megaspore mother cell.
Stage FG1
This is a growing ovule just after generation of the gametophyte via meiosis. The outer (O) and inner (I) integuments are growing up from the chalaza (C) and will soon surround the nucellus (N), which holds the developing embryo sac.
Stage FG1
A closer view of the megaspores after meiosis. The asterisk marks the presumptive functional gametophyte, now at developmental stage FG1. (N) indicates two nonfunctional megaspores that are beginning to degenerate (note the slightly emptier look). Soon they will collapse into the dark material to the right. The chalazal-most megaspore is always selected to be the functional megaspore
Stage FG1
A functional gametophyte at stage FG1 (dark nucleus, center) surrounded by two layers of cells in the nucellus and and the partially completed inner and outer integuments. The section in this image is oriented ~90 degrees relative to the axis of the ovule compared to the previous image.
Stage FG1 - Electron Micrograph
This is a stage FG1 embryo sac in an ovule viewed by electron microscopy. The two dark masses above the embryo sac are the degenerate remains of two of the non-functional female gametophytes.
Stage FG1 - Electron Micrograph
Here the FG1 embryo sac is outlined in black and its large is nucleus is outlined in white. M = mitochondria. Degenerate = degenerate remains of nonfunctional female gametophytes. H = Holes in resin left by imperfections in the embedding process.
Stage FG3
Stage FG3: The growth of the central vacuole has pushed the two nuclei to opposite ends of the syncytium.
Stage FG4
Stage FG4: Two syncytial nuclei are visible adjacent to each other in the plane of sectioning, indicating that the second round of mitosis has occurred.
Stage FG4 - PI staining
A stage FG4 embryo sac in an ovule visualized by a different method: propidium iodide staining and confocal microscopy. The sycncytium of the embryo sac looks smoother than the surrounding maternal cells, and the nuclei are larger.
Stage FG5
One end of a stage FG5 embryo sac just prior to cellularization. Three syncytial nuclei are visible in this section.
Stage FG5 - Electron Micrograph
An EM image of two nuclei in a syncytial embryo sac at stage FG5 just prior to cellularization. Note the absence of any sort of cell wall or plasma membrane separating the two nuclei. This is the same cell shown in the previous image.
Stage FG5 or FG6
An embryo sac after cellularization, stage FG5 or FG6. individual cells are visible at both ends of the gametophyte, separated by the large vacuole that is now contained within the central cell.
All these images are of Arabidopsis pistils that were high pressure frozen, freeze substituted with 2% osmium tetraoxide in acetone and then embedded in epon resin as described in Otegui et al. 2001 Plant Cell 13: 2033–2051. 1 µm thin sections were cut with a microtome and stained in toluidine blue to visualize the different stages of ovule development and as a prelude to cutting 90 nm ultrathin sections for electron microscopy. I did this work in the lab of Sebastian Bednarek in the Department of Biochemistry at the University of Wisconsin, Madison while I was a graduate student there. I also have images of Arabidopsis pollen development.
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