Studying cell fate and cellular organization during plant reproduction
A key step of sexual plant reproduction is sporogenesis, which mediates the transition from the sporophyte to the generation of a gametophyte. Drs. Baroux, Schmidt, and Grossniklaus from the University of Zurich used Imaris image analysis software to help them understand the mechanisms involved in sporogenesis, revealing information that could help in engineering crops with desirable genotypes.
In plants, the first cell of the female germline, called the megaspore mother cell (MMC), undergoes meiosis to form a tetrad of reduced megaspores. The female gametophyte, which contains the female gametes, forms through mitotic divisions from one of these megaspores. After double fertilization of the two gametes, they form the embryo and the endosperm found inside the developing seed.
Drs. Baroux, Schmidt, and Grossniklaus used Imaris to perform quantitative analyses of cell identity markers at the single-cell level and in whole-mount plant tissues. They conducted 3-dimensional image reconstruction in Surpass mode, in silico object isolation using contour surfaces, and fluorescent signal quantification using the software’s statistical tools.
“Imaris allows us an unprecedented qualitative and quantitative resolution in understanding cell fate determination and cellular organization during plant reproduction,” Dr. Baroux says. “Imaris is an essential tool in our research.”
The researchers first analyzed cell-type specific transcriptional profiles. They identified the RNA helicase MNEME (MEM) as predominantly expressed in the MMC and discovered that MEM restricts germline fate to one cell per ovule. More than one MMC-like cell forms in mem mutant plants, leading to the development of additional female gametophytes. The researchers used Imaris to determine the ploidy level of the additional gametophyte(s) in mem mutants.
The investigators used H2B-YFP as a fluorescent marker under the control of the gametophytic AKV-promoter. They took a series of confocal stacks of mem mutant and wild-type cells and then reconstructed images in 3D mode. They used the Imaris surface tool to draw contour surfaces around each nucleus, which expressed YFP. They also quantified the fluorescence intensities in the fluorescence channel by obtaining values for the sum of voxel intensities in the 3D-reconstructed images.
“Imaris is highly efficient for the precise measurement of intensity values for the selected 3D volume elements,” Dr. Schmidt says. “Its capability to measure fluorescence intensity for a selected region of 3D objects, combined with its speed and precision, was most useful for this study.”
The Imaris analysis revealed that the gametophytes in the mem mutants had a higher ploidy level than the wild-type cells, indicating that they were formed from a somatic cell, i.e. through an apomictic process. The fluorescent signal intensities were approximately two times higher in the nuclei of the additional gametophytes than in the wild type, demonstrating their unreduced nature.
The investigators continue to use Imaris as they move forward in this research. “We are extensively using Imaris to quantify cellular and chromatin markers in whole-mount reproductive tissues, such as in developing ovules and pollen of Arabidopsis plants,” Dr. Baroux says. “For this, we are also using Imaris for 4D reconstruction and for deriving 4D statistics in object tracking.”
Research paper: Schmidt A, Wuest SE, Vijverberg K, Baroux C, Kleen D, et al. (2011) Transcriptome Analysis of the Arabidopsis Megaspore Mother Cell Uncovers the Importance of RNA Helicases for Plant Germline Development. PLoS Biol 9(9): e1001155. doi:10.1371/journal.pbio.1001155