Comparison of the wild-type and ems1 mutant anther development. The microscope images show the upper right lobe of the anther.
(A) Wild-type anther at stage 4 with the epidermis, endothecium, middle layer, and precursors of tapetal cells; cells of a particular layer are not formed simultaneously.
(B) Stage-4 mutant anther with a structure similar to the wild-type in (A)
(E) Wild-type anther at late state 5 showing the epidermis, endothecium, middle layer, tapetal layer and microsporocytes.
(F) Mutant anther at late stage 5 with excess microsporocytes and a more obvious absence of the tapetal layer.
31 July 2002 -- A new gene shown to be essential for pollen production in flowering plants has been discovered by scientists at Penn State University. A paper describing the team's discovery of the gene, whose activity they found is necessary for the formation of cells required for pollen production, will be published in the 1 August 2002 issue of the journal Genes and Development.
"This research is the first indication that a specific kind of protein known as a receptor-linked protein kinase, which results from the gene, is important for pollen production in the anther--the male reproductive organ in flowers," says Hong Ma, professor of biology and the leader of the research team that made the discovery. "Plant breeders eventually may be able to use this information to control pollination in important agricultural crops such as wheat, rice, and soybeans, where such control previously has not been feasible."
To identify the gene, the team worked with the Arabidopsis weed--a plant widely used in research laboratories and one of the few whose DNA has been so thoroughly studied that scientists know the order, or sequence, of essentially all its component nucleotide compounds, which make up the plant's genome. Although they have completely sequenced its genome, scientists are just beginning the process of discovering the function of the plant's 25,000 or so individual genes--the various groups of nucleotides strung end to end along its DNA.
To discover both the gene that is required for pollen development and its specific effect on the plant's anther cells, the researchers first generated a group of plants with different mutations and identified among them one plant with a mutation that disabled its ability to produce pollen. They then observed how the development of the mutant plant's pollen-production cells differed from that of a normal plant and they also used genetic techniques to determine specifically which gene was disabled by the mutation. "We deduce what the gene is doing in the normal plant by looking at what the mutant plant is unable to do," Ma explains.
As they studied the development of pollen grains in the mutant plant, the researchers examined the behavior of cells called microsporocytes, which normally undergo a process called meiosis during which they each divide into four daughter calls called microspores that later develop into pollen grains. The scientists found that meiosis in the mutant plant proceeded completely normally until the very last step, when the cells failed to divide and the pollen grains never formed.
"In normal plants, a shell-like structure in the anther of a flower, called the tapetum, provides a kind of nest where the pollen cells form. The microsporocytes fill the interior of this tapetum nest before they divide to form the microspore cells that develop into pollen grains," Ma explains. "But we found that no cells in the developing anther of our mutant plants ever grew into tapetum cells," he says.
To determine for sure whether the cells they observed were tapetum cells or microspore cells, the researchers tested them for the presence of two molecular markers--one that is known to be active only in tapetum cells and the other that is known to be active only in microsporocyte cells. "These molecular tests indicate that our mutant plants have absolutely no tapetum cells and that they have additional microsporocytes where the tapetum cells normally should be," Ma says. Ma and his team have named the gene "Excess Microsporocytes 1" (EMS1), to indicate its effect in the anther of a flower when this gene is not working properly.
The mutation in the research plants was caused by a gene known as a transposon that has the distinctive ability to jump to different locations along the DNA molecule, inserting itself inside one gene or another. Ma and his team cultivated plants that contained this jumping gene and found one plant that did not produce pollen. Then, using standard molecular techniques, they revealed the identity of the jumping gene's location in that plant--within the gene whose pollen-production function they suspected was being disabled by the presence of the jumping gene. The researchers then allowed the transposon to jump away from the gene, and they found that the plant's ability to produce pollen was restored to normal, proving that the jumping gene's former location was within a gene that is essential for pollen production.
The scientists then went on to determine the nature of the protein that the cellular machinery produces from the code contained in the EMS1 gene, and to speculate on its function. They found that the EMS1 protein has three parts--one part that extends outside the surface of the cell membrane, another part that extends into the interior of the cell, and a third part that connects the two and is embedded in the cell membrane. "This protein has the characteristics of a fairly common group of proteins found in plants, known as receptor-kinase proteins, which have a receptor extending outside the cell membrane that binds to certain molecules there," Ma explains. The change produced by this coupling triggers the interior portion of the protein--called a protein kinase--to catalyze reactions between phosphate groups and other proteins inside the cell," Ma says.
"Based on our research and the well-known function of similar proteins in animal cells, we speculate that the EMS1 protein that results from the EMS1 gene we discovered behaves like a protein kinase in the developing anther cells to promote the formation of the tapetum layer that appears to be critically important for the formation of pollen grains," Ma explains. He also speculates that these cells are programmed to become microsporocytes unless the signal from the EMS1 protein tells them to become tapetum cells, and that the formation of microspores requires the presence of the tapetum shell around the microsporocytes, which cannot divide without it. "If our speculations are confirmed, this is the first gene coding for a receptor kinase found to control plant fertility specifically by controlling the development of the anther cells," Ma says.
Ma expects that genes of this type are likely to be found in important crop plants, and that male-sterile plants might possibly be made by inactivating the appropriate gene. "Now that we have identified this gene and its function, plant breeders can disable it with more stable methods that do not involve using genes that can jump around," he says.
One advantage of male-sterile plants is in the production of hybrid varieties of some species that currently are difficult and expensive to produce in quantity. In many crop plants, the male and female organs are packed closely together into the same tiny flower, plus some plants like rice and grains produce only one seed per flower. "For rice, wheat, soybeans, and most other crop plants, it is not economically feasible to produce large quantities of hybrid seeds by removing the tiny anthers," Ma explains. If these plants were male sterile, however, it would not be necessary to remove the anthers in order to control the pollination process.
Another potential use of male-sterile plants is in crops that are valuable mostly for their flowers and not for their seeds or fruits, such as ornamental plants. "If we can limit the production of pollen, and the resulting production of seeds and fruits, we may allow the plant to put all its resources into making more robust or more numerous flowers or perhaps extending the length of the flowering season," Ma says.
Crop plants that are genetically engineered to be male sterile also could be useful in controlling the spread of genes from genetically modified organisms into wild plants. "Male sterility could prevent the spread of a genetically modified plant because, since it would not produce pollen, it could not cross-pollinate any of its wild relatives," Ma explains.
In addition to Ma, other members of the research team include Da-Zhong Zhao, a postdoctoral associate; Guan-Fang Wang, a graduate student; and Brooke Speal, an undergraduate student. This research was supported by the National Science Foundation, the U. S. Department of Agriculture, the National Institutes of Health, and Penn State.
Click here to download a high resolution image of the combined microscope images above.