Russell Laboratory Research Page

This is an introductory webpage describing our approaches toward understanding sexual plant reproduction. We are interested in the control of sexual reproduction in angiosperms. In particular, we have been interested in the transcriptome of male and female gametes of rice and Plumbago, which express typical and preferential fertilization, respectively. Control of expression through epigenetics is a focus as this influences the initial chromatin state of the zygote and young embryo.


Maternal to Zygotic Transition. Our current work on rice focuses on the transcriptome of the gametes and zygote at specific points in the timetable around fertilization using RNA-Seq to provide whole transcriptomic data. This is part of a collaboration with the laboratory of Professor V. Sundaresan of University of California-Davis. To date, we have worked out all of the needed technologies to conduct controlled pollinations of rice plants and to be able to collect fertilized zygotes at specific landmark times during the post-pollination development of rice. All isolation are conducted using living ovaries and rapid dissection of component cells, which are then frozen in liquid nitrogen and retained at -80°C until processing.

Male and Female Gametes Microarray results. Prior to the RNA-Seq approach, we used the 57K microarray of Affymetrix to characterize male and female genes expressed. The data is archive on GEO at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17002.

Preferential Fertilization. In Plumbago zeylanica the two sperm cells that participate in double fertilization are different and have different predetermined fates. One important structural difference between these sperm cells is the one of them is physically associated with the nucleus of the pollen grain, known as the vegetative nucleus (or VN). The sperm cell associated with the VN is known as the Svn. The other sperm cell, in turn, is connected with the prior Svn, but is unassociated with the VN, so it has been termed the Sua. The former fertilizes the central cell and its sperm nucleus, together with the female polar nuclei, establishes the primary endosperm nucleus and the nutritive endosperm. The Sua fuses with the egg cell and forms the zygote, which divides to form the embryo and ultimately the new seedling. The control of this phenomenon is our principal interest.

We have used a number of methods to characterize this. First, we collected ESTs (expressed sequence tags) from the two different populations of sperm cells found in the pollen of Plumbago zeylanica. Additional structural distinctions between the cells are numerous, but one of the most significant is that the Svn contains many mitochondria (ave 252) but few plastids (usually none, but up to two have been observed). In contrast, the Sua contains numerous plastids (ave. 26) but only 20% of the number of mitochondria (ave. 52). The sperm cells preferentially fuse in the embryo sac and follow the pattern above in over 95% of observations. The technology of sperm cell collection is shown in Figure 1. In wind pollinated plants that produce over several grams of pollen per plant (e.g. maize), it is possible to collect cells en masse using FACS (fluorescence activated cell sorting). Plumbago is a limited model for mass collection, however, because it is insect pollinated. Each flower contains five anthers that under ideal conditions produce 256 pollen grains. This limits collection amount, but our collection method, because it is based on manual selection is more accurate at discriminating cell types. We select sperm cells from associated cell pairs. Cells that cannot be discriminated, if any, are omitted. Cell collections are frozen every 10 to 15 minutes and quickly frozen in liquid nitrogen. Thus, each collection is to a specific cell type. Collections are subsequently pooled as needed.

These results indicate that sperm cells of flowering plants are intact cells with diverse messenger RNA populations. A portion of them could be successfully BLASTed to obtain putative protein sequences. These potential homologs can be categorized into different functional classes. For the Sua, 147 sequences were identified that are involved in information replication, storage, cellular process and metabolism pathways, but most sequences of the Sua could not be classified, including 229 hypothetical and unknown proteins. The other 340 sequences had no database match. This indicates the limitation of current databases, as there are only four flowering plants for which sperm cell EST sequences are currently available. That so too few plants have been sampled for this tissue to be meaningfully compared for the diversity of genes that may be involved in angiosperm gamete biology and double fertilization. Currently a few common homologs are found among the available sequences. Perhaps this indicates that current coverage is remarkably poor, but an alternative explanation is that sperm cells may be highly variable between maize, rice, tocacco and Plumbago. This also applies when the sample is expanded to include generative cells of lily, which currently represents the best described generative cell library. As stated above, we intend to obtain at least 10,000 high-quality sequences from each sperm cell cDNA library. This will help to establish a representative profile of relative gene expression between the two sperm cells. Enhanced gene sampling using other flowering plant sperm cells will also improve coverage.

Some recent publications (with PDF links):


Here are some further resources:

This and other pages on this site are under construction. Please forgive incomplete coverage of our current work as we do not always update it as often as we might.


This file was last modified on Sunday, 22-Feb-2015 22:26:32 CST 190067