DURHAM, N.C. -- Researchers have created the first detailed map of when and where some 22,000 genes are expressed in each cell of the growing root of the small flowering plant Arabidopsis.
Their achievement, they said, offers biologists not only a new resource for exploring how complex plant tissues develop from a single cell. The analytical technique they used also will give biologists a basic tool for exploring how tissues arise from individual cells in other organisms. Also, said the scientists, the new information will contribute to more sophisticated efforts to genetically improve crop plants.
The researchers, led by Duke University molecular biologist Philip Benfey, published their findings in the December 12, 2003, issue of the journal Science. Besides Benfey, who is professor and chair of biology at Duke, other authors are Kenneth Birnbaum, Dennis Shasha and Jee Jung of New York University, Jean Wang of Duke, and Georgina Lambert and David Galbraith of the University of Arizona. The research was sponsored by the National Science Foundation.
The widely studied Arabidopsis -- a member of the mustard family that includes cabbage and radish -- is considered to be the laboratory mouse of the plant kingdom. The small, prolific flowering plant grows easily and quickly and includes all the biological structures and functions typical of flowering plants.
Particularly important to Benfey and his colleagues is that the Arabidopsis root offers an easily observable and accessible model of the development of complex tissue from a single cell. Unlike the impossibly intricate convolutions and migrations of developing animal tissues, each new cell in the Arabidopsis root arises conveniently from its neighbor. Also, said Benfey, the root has a radial symmetry that makes identification of specific cells easy, and the number of cell types in the developing root is relatively small.
In creating the mosaic of cells in the developing root, myriads of genes switch on and off to regulate the production of proteins that are the workhorse molecules in building cells. According to Benfey, the achievement in mapping this gene activity, or expression, in the multitude of individual cells of the Arabidopsis root constitutes a significant advance from previous analyses.
"This is the first time that anybody has achieved this level of resolution of gene expression on a global basis for any organism," he said. "Other genomic studies, in which whole tissues were ground up and their global gene expression profiles determined, certainly generated much useful information. However, critical information on the mechanisms of development was lost. Development occurs at the single cell level, and there's a dramatic difference from one cell to the next, in terms of its gene expression," said Benfey.
What's more, said Benfey, "We believe this is a proof of principle that shows that similar approaches can be applied to other plant organs and other organisms."
In their mapping studies, Benfey and his colleagues developed techniques to quickly separate and identify the individual cell types in the rapidly developing root. Determination of the identity of specific cells was made possible by marker genes the researchers attached to genes that were characteristic of each type of cell. Those marker genes produced a telltale fluorescent protein when the genes were switched on, which could be used to separate each type of cell.
The researchers used methods pioneered in Galbraith's laboratory at the University of Arizona, including a fluorescence-activated cell sorter, to isolate the different root cells. He said, "It was a nice balance -- we wouldn't have done it without their presenting the research problem, and they couldn't have done it without this technology." Galbraith added, "This work is an example of how collaborative research can lead to great progress." Benfey said, "One of the secrets of success for this work was that from the time the roots are put into the solution to separate the cells to the time they come out of the cell sorter is only about an hour and a half." He added, "The cells are intimately connected to one another and constantly signaling to one another, and if you wait much longer, they begin to change their gene expression."
Once the researchers had separated a given cell type, they used DNA microarrays, or "gene chips," to measure the activity of each of about 22,000 genes in each cell type. These thumbnail-sized gene chips contain a vast array of Arabidopsis genes. When the scientists add mixtures of tagged genetic material from the individual types of Arabidopsis root cells to the chips, the chips indicate which genes are activated. The results of such analyses yielded several surprises, said Benfey.
"To me, one of the most surprising things was that almost half of the 10,000 genes expressed in the root showed dramatic levels of tissue-specific expression," said Benfey. "I would have guessed perhaps 10 to 20 percent of Arabidopsis genes would have been so expressed." Thus, he said, the plant apparently uses a very large fraction of its genome in the process of development.
Benfey and his colleagues also detected distinct clusters of gene expression, in which different types of root cells tended to express particular sets of genes. Such patterns, he said, will prove valuable to biologists seeking to better understand the genetic machinery of development. And, the scientists detected clustering of genes with similar expression patterns on the plant chromosomes.
Besides aiding in basic understanding of plant development, the new information about gene expression could aid development of methods to improve crop plants, said Benfey.
"A key to understanding development of plant tissues and organs is determining how whole networks of genes are regulated during development," said Benfey. "In applying systems biology to agriculture, we are moving away from just altering one or two genes at a time. We are moving toward altering broad traits, such as seed production, flowering or oil quality, through the use of small molecules that affect regulation of these networks. The techniques we have developed of global measurement of gene expression represent a first step in the process of understanding such networks."
Thus, said Benfey, he and his colleagues are proceeding to manipulate genes involved in the expression patterns they have discovered, to explore the effects on the plants' biological machinery.
Three years ago, following an international effort, Arabidopsis became the first plant to have its genome sequence completed. The National Science Foundation, a key funder of the sequencing effort, then launched "Arabidopsis 2010," a multinational program to determine the function of the all of the plant's genes by the end of this decade.
According to Joanne Tornow, a program director in NSF's Division of Molecular and Cellular Biosciences, "The creation of the root map is a terrific advance forward. This new approach allows a global analysis of gene expression in specific cell types in a complex tissue over a gradient of developmental time. This allows the analysis of the regulation of gene expression at a much finer resolution."
The process should work with plant tissues beyond the root, although there it may be more difficult to observe changes in gene expression over developmental time, said Tornow.
"This lays the groundwork for looking at how various biological pathways interlink in transcriptional networks," she said. "There are still thousands of genes in Arabidopsis, about the function of which we know almost nothing. By knowing when a gene is expressed and where it is expressed, we get clues about the processes it is involved with and potentially its function as well."
Materials provided by Duke University. Note: Content may be edited for style and length.
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