No shrinking violet
by Cherie Winner | © Washington State University
There's a buzz in the world of botanical science. Led by Anthony Trewavas, a highly-regarded scientist at the University of Edinburgh and a member of Great Britain's Royal Academy, researchers all over the world are suggesting that plants are more than a leafy backdrop for Earth's more active and interesting residents. The buzz is that plants communicate. They plan ahead. They remember. They're intelligent.
Are we on the brink of a revolution in biological thought? That depends.
Washington State University psychologist Jay Wright, who studies learning and memory in mammals, wonders what it means to say that a plant is intelligent. Even with respect to animals, he says, "intelligence is in the eye of the beholder."
When sizing up smarts, we often look for intentional actions, goal-directed behaviors we can see and measure. How can plants be intelligent? To the casual observer, plants don't seem to have "behaviors." They seem to passively accept whatever the environment tosses their way.
But researchers at WSU are finding that plants are surprisingly assertive. Based on their findings, a case could be made that the average potted plant is at least as active as the average human couch potato—and a lot smarter about what it consumes and the company it keeps.
Watching their diet
Plants don't "eat," of course, but they do take in energy, in the form of light. They use that energy to convert CO2 and water into carbohydrates. Although plants can't move to a sunnier or shadier spot like a sunbather going for optimal tan, plant physiologist David Kramer says some of them make smaller movements to control their exposure to light. They turn their leaves to intercept more or less light. They even rearrange their internal parts to enhance or diminish their energy intake.
In fact, says Kramer, leaves are so active that they have made it nearly impossible to do certain kinds of experiments for longer than a fraction of a second. For example, when scientists directed narrow beams of light at leaves in order to track changes in the chemicals involved in photosynthetic reactions, the leaves reacted about the same way you or I would to having a bright light shone in our faces. Ultra-short experiments worked fine, but anyone wanting to watch what happened over a span of minutes was out of luck.
"The plants were moving," Kramer says. "They could change the shape of their cells and the chloroplasts, and that scatters the light differently. That's a problem."
His solution was to invent an instrument that scrambles the light before it hits the leaf and efficiently collects the scattered light that comes out the other side. With this machine, movements within the leaf have little effect on the measurements, because the light is already scattered.
Kramer dubbed the instrument Nofospec, for "non-focusing optics flash spectrophotometer," and patented it. Besides the one in his own lab, he has made a Nofospec for colleagues at WSU and the University of California-Davis, and has orders for more from labs in Japan and France.
Kramer says plants have another, even more subtle, way to control how much light energy they feed into the photosynthetic pathway. In weak light, they are incredibly efficient. Their light-gathering apparatus, highly organized protein clusters called antennae, send about 80 percent of the photons striking them into growth and maintenance activities.
In bright light, though, they pull the shades. Instead of funneling the light energy into the photosynthetic reactions, the antennae send up to 90 percent of it back out into the environment as heat.
They have to do that, says Kramer, or risk being bleached and burned by the intense energy concentrated in their chloroplasts.
"Basically, the plants are dealing with explosives," he says. "They need to, to drive all these [photosynthetic] reactions—but if they take in too much, they're going to pay the consequences."
His group is studying how the plant knows to turn its antennae up or down. "People spent decades trying to improve the efficiency of photosynthesis," he says. "And plants are already pretty damn good at it. The key here is matching the regulation to the environment."
And plants are masters at that. They monitor the light striking every bit of leaf surface, and act in a way that takes into account both their need for energy and the risks of overindulgence.
Don't mess with Mum
Plants also do a good job of defending themselves. Their defenses range from crude and always deployed—think thorns—to downright sneaky. How else to describe the fate that befalls certain insect larvae, as told by biochemist Clarence "Bud" Ryan?
"When they chew on the plant, their saliva mixes with the wounded plant tissue, and it gives off volatile [chemicals]," says Ryan. "Predator wasps pick up the smell. Then they come and inject their eggs into the larvae." When the baby wasps hatch, they gnaw on the host larvae from the inside. The larvae, understandably, stop chewing on the plant.
Ryan pioneered the study of another line of defense, which has come to be known as the systemic wound response. A protein chemist by trade, he started in the early 1970s trying to understand how plant protease inhibitors work. Those are chemicals that block the digestive enzymes (proteases) in the gut of any animal that takes a bite out of the plant. Raw potatoes contain protease inhibitors, which is why munching on them can cause severe stomachache.
Ryan found the inhibitors were present in the leaves of potato plants as well—but that some potato plants were loaded, while others had none.
"And I got the idea that maybe they're there because an insect was attacking the plant," he recalls. "So I went out and borrowed some Colorado potato beetles from a friend of mine, and let them chew on potato plants in the greenhouse." A day later, the previously inhibitor-free plants were full of protease inhibitors.
Ryan then repeated the experiment, confining a beetle to a single leaf with a shield of aluminum foil. The next day, leaves on the opposite, uninjured side of the plant contained just as much inhibitor as the bugged leaf. News of an attack at one point on the plant had spread—and caused a response—throughout the entire plant.
"That was a huge discovery, because nobody had ever seen anything like that," he says. The breakthrough opened a whole new field of research, and secured Ryan's election to the National Academy of Sciences in 1986. In 1991, his research team isolated the signal chemical, a small peptide they dubbed systemin. It was the first signal peptide ever found in plants.
Plant protease inhibitors do more than just give bugs an upset tummy: they send a message from the gut to the brain that ruins a bug's appetite.
"It's a satiety thing," Ryan says. "It's telling the insect, 'You're full, you shouldn't eat so much.' At the same time, the insect is starving to death.
And it happens to work the same way on human hunger pangs. Ryan and research associate Greg Pearce devised a large-scale method for producing the inhibitor, which is now being sold by Kemin Industries as a human weight-loss aid called Satise®.
Recent explorations in his lab have revealed the presence of a different signal peptide that activates the plant's defenses against microbial pathogens. In the innate immune response, as it is called, an initial attack by a pathogen prompts nearby cells to make the new peptide, which then travels through the plant's vascular system and causes cells throughout the plant to make substances that fight the pathogen—and which also stimulate the production of more peptide. This "amplification response," as Ryan calls it, allows a plant to respond in a big way to a small attack. If the initial attack is followed by more, the plant will be prepared.
Friends in low places
The innate immune response does a good job of protecting the aboveground parts of the plant. Roots are another story, according to plant pathologists Linda Thomashow and Patricia Okubara.
"Resistance to belowground pathogens is very rare in plants," says Thomashow. "Instead, it seems that plants use as a defense the bacteria on their roots."
Roots, which Okubara describes as "deceptively simple," exist in a sort of ecosystem of their own, known as the rhizosphere. Home to bacterial and fungal cells—several million per spoonful, at least—the rhizosphere is as crowded with opportunity and danger as anything the plant encounters above ground. But it is far from chaotic.
"When I first came to work here, I thought that bacteria on roots were sort of like butter on toast—you know, they were everywhere," says Thomashow. "Not true! They're patchy. They're in the cracks between the cells, they're in places where the surface of the root has been wounded as the root grows through the soil."
Bacteria cluster in these patches, she says, because the plant leaks nutrients at those points—by design, apparently, because the bacteria that come to feast on the leaked goodies produce chemicals that repel more dangerous bacteria and infectious fungi.
"One would think that the amount of carbon being leaked out through the roots was being wasted," says Thomashow. "In fact, it's very unlikely that plants would be so foolish as to waste 20 or more percent of the carbon that they fix. So they feed these bacteria, and the bacteria are sort of a first-line defense."
She says one of the bacterial anti-fungal chemicals, DAPG (2,4-diacetylphloroglucinol), makes the roots of some plants become even more leaky. "So the root responds by feeding the bacteria even more than it did before," she says. "There's a lot of dialog [between plant and microbe]. We call it cross-talk. Molecular dialog."
"The host is actually quite active in this phenomenon," says Okubara. "It's not just sitting back and saying, 'Colonize me. Make all the anti-fungal metabolite you want to.' It actually has a role in the amount of bacteria that grow and the amount of DAPG that accumulates on the root."
Okubara is exploring exactly how root cells respond to DAPG and other substances produced by the bacteria. Using microarrays, microscope slides with tiny grids on which she places DNA probes, she has found several genes that are turned on in root cells exposed to the bacterial substances. She is beginning to identify what the genes code for and what their roles might be.
Thomashow is trying to isolate strains of bacteria that are especially "friendly" to crop plants such as wheat, and develop forms of them that can be used to coat seeds prior to planting. That would allow the helpful microbes to accompany the baby roots when they first poke down into the soil. Such a product would have big advantages for farmers, she says.
"The elegance of the whole system is that the amounts are very small, but they're exactly where they need to be in order to be effective," says Thomashow. "It's the sophistication in nature. And people didn't think of it. Nature was working on this for a long time."
That sounds like a good system, but it raises a problem. Roots are immersed in a Mardi Gras of microbes benign, beneficent, and bellicose. How does the plant tell which is which?
The secret, says molecular biologist B.W. "Joe" Poovaiah, lies in the molecular dialog between root and microbe. He and his research team have decoded a crucial part of the conversation in legumes. Those are the plants, such as peas and beans, that form symbiotic relationships with bacteria that convert atmospheric nitrogen into a form the plant can use to make proteins and other organic compounds.
Here's how the conversation starts: Delicate root hairs that grow from the main roots exude chemicals called flavonoids. Bacteria in the soil are attracted by the flavonoids. They sidle up to the root hairs and, in effect, ask to come in. Poovaiah calls it "knocking on the door."
When helpful, nitrogen-fixing bacteria are trying to establish a relationship with a plant, they knock by secreting a chemical called Nod factor. Harmful bacteria don't secret Nod factor—and the plant knows that.
When a root hair cell receives the Nod message, it begins to move calcium around. Poovaiah's coworkers at the John Innes Centre in the UK showed this with special microscope equipment that allowed them to make a movie in which different levels of calcium appear as different colors: blue for low, red for high. Running in real time, the movie shows a blob of red squishing back and forth inside the root hair cell. It looks like a pixilated image of a beating heart. (To view the movie, see sidebar, bottom.)
"This is not just calcium going in," says Poovaiah. "It's not a flood. It is very rhythmic, coordinated in the intensity and the duration. That's the beauty."
The calcium pulses carry a message. Soon after they begin, the cell turns on specific genes, and the root hair bends around the bacteria like an enfolding arm. Eventually, the bacteria and root hair combine to form a nodule, visible to the naked eye, in which each partner supplies something that benefits the other.
"The door is only open to the bacteria that have the key, that sent in this Nod factor [and caused the calcium pulses]," says Poovaiah. "The enemies cannot create that." If a root hair encounters harmful bacteria, he says, the hair "would just stay there and say, 'I'm not going to talk to you.' That is the mystery: only friends come in. Enemies stay out."
Sathyanarayanan Puthanveettil ('01 Ph.D.), then a student in Poovaiah's lab, worked on one of the proteins involved in this sequence of events. It's a kinase, an enzyme that binds to calcium and then promotes changes in other proteins in the pathway. Poovaiah calls it a "decoder" that interprets the encrypted signal carried by the calcium pulses, and triggers the next steps in the pathway.
Biologists have long known that calcium plays a key role in animal systems. It's important in nerve transmission and muscle action, in addition to its structural role in bones and shells. Over the past 30 years, Poovaiah has established that the mineral is just as important to plant adaptation and survival. His lab has shown that calcium is the major player in a messenger system that helps the plant monitor and respond to more than a dozen environmental variables—functions that, in animals, are performed by the nervous system.
Still, he and Puthanveettil were startled when they realized that a large part of their kinase is very similar to a kinase found in mammalian brains. The mammalian kinase is called a "memory molecule," because it plays a key role in the formation of long-term memories.
"When we cloned the gene for this kinase, we thought, 'There must be something wrong here. It cannot be,'" recalls Poovaiah. "So we went back [and checked again] and we said, 'No, that's the way it is.'"
Other scientists were surprised, too—and impressed. Puthanveettil was recruited to do postdoctoral research at Columbia University in the lab of Eric Kandel, who won the Nobel Prize in Medicine in 2000 for his work on signal processing in the nervous system and the biochemical mechanisms of memory storage.
"I thought I would like to challenge myself in a very complex system—the brain," says Puthanveettil. He is now analyzing kinases and other proteins involved in learning in a marine mollusk called Aplysia.
Poovaiah was invited to present his work on calcium signaling at a conference in Beijing in May 2006. The meeting is hosted by the Society for Plant Neurobiology.
You read that right: plant neurobiology.
That term doesn't make sense to psychologist Wright. Plants don't have nerve cells or nervous systems, after all.
On the other hand, they clearly have a system of biochemical communication between cells, a system that allows a plant to direct its own behavior and interact in specific ways with other organisms. Neurobiologists say that 99 percent of all communication in an animal's brain is chemical, not electrical. Why couldn't plants be doing something similar?
Pressed for an opinion about plant "brains," Poovaiah laughs. "You want the Poovaiah model? Plants don't have one big brain, they have tiny brains everywhere." Control is diffuse rather than centralized. Different parts of the plant direct different aspects of behavior.
The root tip, for example, senses gravity and directs the root to grow downward. Cut off the tip, and the root wanders aimlessly. Replace it, and it heads downward again. Charles Darwin did that experiment in the mid-1800s.
"He said the root tip is like the brain of a small animal, like maybe an earthworm," says Poovaiah. "We do know there's something. They're not as passive as we thought. They do have the ability to sense changes and respond. In that sense, they are intelligent."
There's that word again.
"I think the problem is starting with definitions we can all live with," says psychologist Wright. He thinks it will be difficult to figure out whether plants act in a flexible, problem-solving (i.e., intelligent) way, or whether they simply execute "fixed action patterns" they are genetically programmed to do.
For instance, a plant whose root recognizes and embraces helpful bacteria will thrive better and leave more offspring than plants without that ability; a plant that embraces the wrong kind of bacteria might not live long enough to reproduce at all. A complex behavior that looks intelligent could have arisen and been highly refined through eons of evolutionary pressure.
Poovaiah is intrigued by the current speculation about plant "intelligence," but for now, he is content to learn more about how calcium signals work, and what they reveal about the inner life of plants.
"It was private," he says with a sly smile. "But now we have opened the door."
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