In the Friar's Footsteps

Wagon trains were rumbling through the American West on their way to the Oregon Territory in 1853 when, halfway around the world an
inquisitive young friar started conducting research at a European
monastery dedicated to the study of theology, philosophy and the natural sciences. Over the next couple of decades, as pioneers from the East and Midwest settled western Oregon's Willamette Valley, the friar made far-reaching discoveries, though they went pretty much unnoticed during his lifetime.

Recently scientists at Oregon State University, located in the heart of the Willamette Valley, added significantly to the friar's story. William Proebsting and David Martin, researchers in OSU's Department of Horticulture, isolated and cloned the famous dwarfing gene that Gregor Mendel noted in his experiments at the monastery.

Since Mendel's time, the fields of genetics, plant physiology and pathology, chemistry, and molecular biology have contributed separate
strands toward the knowledge of how this dwarfing gene works.
Proebsting and Martin's paper, published in the Proceedings of the
National Academy of Sciences, punctuated this long series of inquiries by showing precisely the genetic change that dramatically alters plant growth. People around the globe may someday benefit from agricultural practices that use space-efficient dwarf plants or trees grown as a result of this research.

Illustration: Tom Weeks

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A walk to OSU's Agriculture and Life Sciences Building, a trip up the elevator to the fourth floor, a trek down a long hallway and through a door, a few steps past the refrigerator and a right turn at the freezer lead to an immaculate laboratory. It is here that Proebsting, a professor of horticulture, and Martin, a research assistant, did their gene-cloning project.

Because Proebsting's work with the dwarfing gene takes place in a
classical science context, he begins an explanation of what he and Martin did by referring to Mendel. There was a "long period of progress, starting with Mendel, who uncovered the relationship of the control of plant features by certain characters," says Proebsting. "He [Mendel] didn't have much of an idea of what they were. We now know they were genes. He could tell that there were two forms of a gene. There was a dominant form and a recessive form. One form dominated the other."

Initially, Johann Gregor Mendel experimented with flowers in the gardens of his Augustinian monastery in Brno, Austria. His desire to develop new color combinations led him to tests with hybridization or cross-breeding. Mendel chose the common, easy-to-grow garden pea for this cross-breeding experimentation. He performed hundreds of experiments on an estimated 25,000 pea plants.

Mendel wasn't the first scientist to carefully observe the lineage of plants. People had been making observations, however unsystematic, since the beginnings of agriculture. In the 18th and 19th centuries, biologists were becoming increasingly eager to figure out the paths of inheritance in plants and animals. A typical theory was that there were particles in each parent's body; these particles blended in the offspring. That was about as far as things had gone. The fact that chromosomes with genes made of DNA govern inheritance wasn't known until the 1950s.

However, Mendel made a breakthrough in the understanding of inheritance. His pea plants exhibited what are known as Mendelian "traits" (there are seven). Some ripe seeds were smooth, others wrinkled. Some unripe pods were green, others yellow. And some stems were tall, others dwarf. Mendel's systematic notation of what happened to generation after generation of pea plants was laid out in a famous paper published in 1860.

Horticulture professor Bill Proebsting in an OSU greenhouse. He and colleague David Martin used pea plants like these to clone for the first time a gene discovered in the 1800s by Gregor Mendel. Photo: Bob Rost

Still, as Proebsting points out, Mendel was not hailed during his lifetime. "He'd published his paper, but it didn't make a strong impression on anyone. He'd done a lot of things as a teacher and scientist. But when he died, he believed that his legacy would be in meteorology." The man who would come to be known as the "father of genetics" was forgotten, for the time being.

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The second chapter of the story leading to Proebsting's work started in Japan around the turn of the century. "Plant pathologists were studying a disease of rice," he explains. "This disease caused the rice to grow tall and fall over. It turned out that the fungus made plant hormones called gibberellins (GAs). We now know that these are the plant hormones that control plant height and other factors. The Japanese were able to identify this compound."

The scientific question then became "Are there gibberellins in plants as well as in fungi?" The answer was "Yes." World War II put a halt to most gibberellin research. However, there was "an explosion of interest in gibberellins after the war. Research went on in the United States and Great Britain, mainly because they still had the scientific establishment in place," Proebsting explains.

Scientists were intrigued to find that the hormones, in tiny amounts, had enormous effect on plant growth and other factors. GAs are also important because they're tied to the way plants respond to their environment. Proebsting says, "Environment controls plant growth and one of the factors mediating this is the action of gibberellins."

Proebsting grows animated talking about the discovery of gibberellins. Here at last were some good clues about how plants work. Proebsting comes from a line of people who cared about just that. His grandfather was a horticulture professor at the University of California at Davis; his father was a horticulture professor at Washington State University. And Proebsting the Youngest had followed their lead by becoming a botany major at the University of Washington, then a graduate student in plant physiology. You could say that love of plants is in his family's genes.

"Here we come back to the Mendel story," says Proebsting. When Mendel bred a tall pea plant with a dwarf pea plant, the first-generation offspring were always tall. He questioned where the dwarf variation "went." Mendel found that, when the second generation children pea plants were bred, the variation would appear again. It hadn't disappeared at all; it has just been hidden in the previous generation. His trailblazing paper detailing a system of dominant and recessive genes has earned him, since his rediscovery in the 20th century, the stature he holds in science.

Scientists had early on associated the gibberellins found in plants with the tall/dwarf trait that Mendel had observed. A Mendelian trait shows up in a very simple form, either in one variation or the other. The plant's stem length would be tall or dwarf, with no variations.

In the approximately 50 years since World War II, 110 gibberellins have been discovered, yielding leaps in understanding about plant physiology but also posing questions, Proebsting says. Intensive gibberellin research goes on all over the world, much of it centering around Arabidopsis, a small plant from the mustard family that is being used as a model species for gene research. Gibberellins have "complex biosynthetic pathways," according to Proebsting.

David Martin prepares DNA for electrophoresis, which uses ultraviolet light and electricity to pinpoint genes. He and Bill Proebsting used this process in cloning the gene that causes dwarfing in plants. Photo: Bob Rost

After gibberellins were discovered in fungi and then plants, it was another 40 years before scientists found which gibberellins occur in the pea. As when Mendel used it in his experiments, the pea is an ideal species to study. The discovery that a certain gibberellin added to a dwarf pea would make it tall was a significant advance. It wasn't until the early 1980s that scientists learned that GA1 is the single active GA in pea. GA1 production turned out to be controlled by the gene that Mendel had recognized as the dwarfing gene. "So this is important for controlling plant stature," says Proebsting.

Here's where Proebsting and Martin's work kicks into full gear.
Proebsting has expertise in gibberellin research dating back to his
graduate study at Cornell University. Martin's expertise in molecular
biology comes from his master's work at Purdue University. In
preparation for the complex work of isolating and cloning a gene, the two men spent months studying the genetic structure of the pea plant and other species. They used the information that's available from other plant species, particularly Arabidopsis, to infer what the structure of the gene may be. They focused attention on the two rows of "letters" on the gene that encode genetic information. As Proebsting explains, just as we read alphabetic letters to gain information, the cell's machinery reads these letters and decodes genetic information. A change in a letter is a mutation, which may or may not be biochemically significant.

In the case of Proebsting's experiments, the mutation he had noted was very significant. One letter of the genetic sequence was different. This change prevented formation of GA1 and produced a dwarf plant. Proebsting and Martin had uncovered Mendel's dwarfing gene.

The two men then spent months hard at work in the laboratory before they successfully cloned the gene. The physical work takes place in a test tube about the size of a small match book. At the bottom of the test tube is a "puddle"- a few drops-of water and enzymes, for this process occurs at the molecular level. An enzymatic reaction yields a fragment of the gene. Martin works with this fragment, cutting its DNA at a specific site using what Proebsting calls "molecular scissors." Martin oversees the delicate work of inserting the fragment into a medium so that it can grow. The result of the cloning is billions of copies of the gene.

Why is the cloning of this particular gene significant to agriculture? Scientists and farmers have long recognized the advantages of smaller plants. Many dwarf species have been bred by conventional methods, many to great success. Bush green beans, for example, are now more widely seen than pole green beans. Density of planting is, perhaps, the most obvious advantage. Many more dwarf than tall plants can be grown on the same acreage. Further, while dwarf plants have shorter stems, they're aren't less productive. Dwarf plants may possess the same useable matter as tall plants.

Shorter plants also are less likely to droop than tall ones, which keeps them off the ground and possibly prevents rot or insect problems. Greater air circulation around the smaller plants may contribute to overall plant health.

The pea plant on the right expressed the famous dwarfing gene first identified by Gregor Mendel, the "father of genetics." Photo: Bob Rost

Oregon fruit tree growers may have a special interest in the potential of dwarfing genes. Dwarf fruit trees would reach full productivity quickly. As yet it's been difficult to impossible to develop dwarf fruit trees, other than apple, by conventional methods. Genetic engineering may be one approach to developing dwarf trees.

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An OSU greenhouse on the west side of campus holds dozens of dwarf
and tall pea plants at the ready to be used in Proebsting's and Martin's research. Researchers all over the world will use the information learned from Proebsting's work to further their understanding of plant genetics and broaden their search for genes.

Classical scientists and nature-loving non-professionals are one and the same in this science story. Even more important, in Proebsting's view, is that this work done at Oregon State University gives people a moment to reflect on Mendel's achievement and the knowledge gained since his time.