What they’re finding could ultimately be used by farmers to control the timing of harvests, to give florists flowers that hold tight to their petals or even, as one British reporter suggested to Walker, to grow Christmas trees that wouldn’t drop their needles. “That impressed my siblings, two sisters and a brother, who aren’t scientists,” Walker says. “They had no idea what I did.”
His work also has impressed his colleagues in the field.
“John is a pioneer,” said Frans Tax, a plant biologist at the University of Arizona. “His research is an elegant combination of genetics and biochemistry. Because these genes are found in all plants, manipulation in crops to regulate abscission could be fairly straightforward.”
Before following Walker on his path to discovery, let’s review for a moment the case file on plant abscission. The autumn turning of leaves has always been part of the human experience, an activity that has attracted at least as much interest from poets as from scientists. When Walker does a PowerPoint presentation of his work he includes a quotation from Emily Bronte:
“That just about says it all,” Walker says.
Among deciduous trees, the kind that Bronte wrote about, abscission is used to conserve energy. Leaves change color and fall from trees as days shorten with approaching winter. Nutrients from the leaves are drawn back into the tree and stored for the next growing season. If the leaves were to stay on the trees through the winter, the chances are good they would freeze and die and the energy they held would be lost.
But there are other reasons why plants use abscission.
A tree will drop its fruit in order to disperse its seeds. Or a plant may release its diseased leaves to keep the rest of the organism from becoming infected.
“All of these are programmed in the plant’s development or by changes in the environment,” Walker says. Plants come with predetermined “abscission zones,” places where a petal, for example, can safely drop away without injuring the plant.
Walker reaches for a houseplant on the windowsill of his office. He deftly picks off a leaf from the base of its stem. It comes off easily from the vine, right along its abscission zone. Then he rips a piece off the top of another leaf. It leaves a jagged tear. No abscission zone there.
“Abscission zones are a very specialized set of cells. You’re not ripping cells apart; you’re causing separation between cells,” Walker says.
Abscission zones are formed as plants develop. Like all plant cells, cells in abscission zones stick tightly to each other. But when the abscission process starts, enzymes are formed that dissolve the “glue” holding the cells together.
After the leaf or petal falls, abscission zone cells remaining on the plant may form scar tissue to protect it from disease and keep it from losing moisture. “The plants have evolved this for a reason,” Walker says. But what they do naturally and what people want them to do may be two contrary things. “Wheat, corn, soybeans, citrus: we grow them in a very different way from how they evolved,” he says. “So they do things that we may not want them to do.”
For example, when soybeans are stressed by drought, abscission is triggered and the plants naturally drop their fruit, even though the farmer may be ready to irrigate. When agricultural plants become infected, a farmer may be able to spray them. “But the plant doesn’t know you can do that,” Walker says, so it drops its leaves to keep the disease from spreading.
While many plants engage in abscission, to get to the root of the process, Walker is focusing his investigation on a single perpetrator: Arabidopsis thaliana, a favorite of plant scientists.
A selection of Walker's Arabidopsis plants growing in an MU greenhouse.
“It’s the fruit fly of plants,” Walker exclaims. “It’s the lab mouse.”
Arabidopsis is related to the mustard plant and has seeds as tiny as grains of dust. The plants themselves are so small they can be grown abundantly indoors. “So instead of having fields, we can have growth chambers.” And it grows quickly, taking just six weeks to mature. “We can have multiple crops a year.”
Arabidopsis was the first plant to have its whole genome sequenced; it has about 30,000 genes in all. And, as with fruit flies, genetic variants are readily available.
“There’s a worldwide community that works on them and they share things, so there are a lot of resources out there,” Walker says. “If I am studying a gene I can go on the web and order a mutation of that gene.”
MU has a long history with Arabidopsis. George P. Redei, a geneticist and professor emeritus at MU, brought its seeds to MU when he immigrated here after World War II (as recounted in Illumination’s Spring/Summer 2001 edition).
“Missouri has been a hotbed of plant genetics for a long time,” Walker says. The MU husband and wife team of Ernie and Lotti Sears bred domesticated wheat with the rust resistance of wild wheat. And Barbara McClintock, who was awarded the Nobel Prize for the discovery of the “jumping gene” phenomenon —genes that can “jump” into random areas of a chromosome — also worked at the University.
Walker points through a window to a nearby complex of greenhouses. He has one row of the greenhouse, a strip about 20 feet long by 72 feet wide. Inside it grow about 100,000 Arabidopsis plants.
Walker is also cultivating hundreds of thousands of additional plants. Pallets of them fill incubator-like machines called growth chambers and climate-controlled rooms where the plants often get 16 hours of light each day.
“We grow them as fast as we can, so we use long days,” he says. Walker enters one of the rooms. It’s about the size of a large walk-in closet. It’s warm and bright and has the earthy smell of rich soil.
Row after row of Arabidopsis plants line the shelves. Like dandelions, the plants have leaves that lie low to the ground. Their stalks rise about five inches, although when fully grown, they can be a couple of feet tall.
Arabidopsis flowers have tiny white petals. And it is here that Walker’s detective work into the genetics of abscission begins.
The petals of wild Arabidopsis plants have abscission zones. The petals drop after the plant’s seed pod is fertilized by pollen. Walker flicks a couple of plants with his finger and the petals fall.
But in a mutated variety of Arabidopsis, the petals stay firmly in place. “I can flick them all day long and they never fall off,” Walker says.
These plants provided the clues to tie the abscission process to certain genes that Walker already had identified as suspects. Scientists have long known that different signals, such as pollination or fewer hours of daylight, trigger the production of enzymes that dissolve the bonds between cells in the abscission zone.
“But there’s always been this big, black box between the [triggering] signal and the event, the pathway the signal travels,” Walker says. “We’ve identified a lot of the genetic components in that pathway.”
Genes communicate the signal by producing proteins called enzymes that act on receptor genes that then turn on more enzymes. “What you have is a cascade of enzymes that activate enzymes that activate other enzymes,” Walker says. “It’s the proteins talking to the proteins.”
But out of the 30,000 genes in Arabidopsis, which ones are involved in abscission? “We knew what all the genes in Arabidopsis looked like, so we could make a guess at what they do,” Walker said. “We let the plant tell us.”
Back in 2009, Walker and his Interdisciplinary Plant Group colleague Shuqun Zhang found certain genes that expressed proteins that were concentrated exclusively in the abscission zones of Arabidopsis petals. In the mutated plants that don’t lose their petals, these genes no longer produce the proteins.
So far, Walker and his group have linked four genes that make up the midsection of the abscission pathway. “We determined that these genes all play together,” he says.
The linkages they identified are based on inferences drawn from the differences between the normal and mutant plants. To prove that these genes are involved, the MU scientists will have to demonstrate that there are direct physical interactions between the proteins. That bit of biochemistry is technically difficult, Walker says. “But it’s necessary to prove the theory. That’s how science works.”
Walker and Zhang also hypothesized that there are several more genes involved earlier on with creating and transmitting the initial signal for abscission and, later on, with the actual ungluing of the abscission-zone cells.
To find those genes, Walker and his team have continued looking for more Arabidopsis mutations that modify the abscission process. He is currently working on that project with a graduate student and a postdoctoral fellow. Their goal, eventually, is to determine the entire genetic and biochemical chain of abscission.
One of these team members, post-doc O. Rahul Patharkar, recently made such a discovery, one that Walker has called a “tour de force” in abscission research.
Patharkar looked at a gene called HAESA that was already known to promote abscission. HAESA cranks up its activity by 27 times in just the day or two before abscission starts and plants drop their petals.
“A lot of gene expression changes before abscission,” Patharkar said. “We didn’t know how it was happening.”
Patharkar found how two proteins are involved with determining how active HAESA becomes. One protein, an inhibitor, keeps the gene at bay. When it’s time for abscission to start, another protein switches that inhibitor off. At first, the gene “barely comes on,” he says. But gradually it gains steam, promoting the production of more of the HAESA-activating protein and deactivates greater amounts of the inhibiting protein.
With less inhibiting protein, HAESA becomes even more active, which, in turn, leads to more of the activating protein and even less inhibiting protein.
“It’s not a linear pathway; it’s a feedback loop,” Patharkar said. “It works like a turbo booster for a car.”
Knowing how this process works may, one day, be useful in agriculture, Patharkar said. Soybeans, for example, are quick to drop their flowers at the first signs of drought. If farmers could stop that from happening, they could increase their yields, he said.
On the other hand, apple farmers want to limit the number of flowers on their trees; fewer flowers allow trees to grow larger apples. More sophisticated knowledge of abscission may lead to better-targeted chemicals for pruning flowers from the trees, Patharkar said.
Abscission In Action
Red stains reveal the "abscission zone" on a leaf. The scissor cells depicted mark the boundary between the branch (left) and the leaf stalk.
Abscission genes appear to be conserved among plants, so knowledge about Arabidopsis could be applied broadly. Once the genes are known in one plant, scientists can look for similar genes in other plants.
“There might be some differences in the details, but the fundamentals are going to be there,” Walker says. “These genes have been around for a long, long time and if they weren’t important, evolution would have gotten rid of them.”
Ultimately, he adds, a more complete understanding of the genetics of abscission could increase scientists' general understanding of how genes perceive and respond to chemical signals. Such a breakthrough could lead to important insights for both plant and human biological studies.
Whatever scientists find, it will take much less time than it would have when Walker started his career more than 20 years ago. He marvels at how technology has sped up research. Instead of looking at one gene at a time, whole genomes are open to study.
“We’re making discoveries a lot faster then we used to,” he says. Walker got his first exposure to the science of biology while growing up in Phoenix, Arizona. For many years, he spent his summers working for his father, a veterinarian. He recalls it as a positive experience, but he wasn't tempted to follow in his father's footsteps.
“It pretty well convinced me I didn’t want to be a vet or a doctor,” he says.
As an undergraduate at Arizona State University, however, Walker worked in the laboratory of a plant biologist and became fascinated with the biochemistry of plants. Later, as a graduate student at the University of Georgia, he became interested in genetic signaling. After earning his doctorate in 1985, Walker went to Australia to work with a leading research group studying gene transcription. In 1987, Walker joined the MU faculty.
Soon after coming to the University he discovered receptor genes in plants, a finding that opened up a whole new realm of research. “These looked like receptors in us, like insulin receptors,” Walker says. “People really didn’t think they were in plants. They didn’t think plants used protein signals.”
Classical genetic research starts with a mutation in an organism and then looks for the gene that is involved. Walker employed what he calls “reverse genetics” to discover what process these receptor genes mediated. First he looked for where the genes were expressed and located that activity in the abscission zones. Then, the mutated plants that didn’t lose their petals showed him what happens when the genes are disabled.
“The nice thing is putting a lot of pieces together,” he says. “People want to be able to control those things. And the more you know about the process, the more opportunity you have,” Walker says.
But for Walker, the practical applications aren’t the only motivation for drilling into the genetics of plants. The biggest thrill by far comes from tracking down new pieces of scientific evidence and making the deductions that tie them together.
“It’s discovery,” Walker says. “That’s one of the things I’ve always loved about science: discovering new stuff. And at a university, we can share it with students.”