The Green Question

Elaine M. Tietjen
March 01, 1996

"Here's a silly question," says Jack Schultz: "Why does the world remain green? Why don't the insects eat everything every year?"

Schultz, an entomologist who finds himself working on plants as often as on insects, likes to pose apparently simple questions — perhaps because he's been led into many an intriguing problem that way. Now he races headlong into the puzzle, pulling me after him in a sort of intellectual tag.

green forest

"Think about this," he says. "An oak tree doesn't reproduce before the age of 20. Every time it reproduces, the mortality of its offspring runs in excess of 99 percent. It sits there for 300 years and during that time, some of the insects that eat it go through 600 or 1,000 generations. Given the relatively slow reproduction rate of the tree and the ease with which insects appear to adapt to new circumstances, you would think there's more than enough time for insects to evolve the ability to eat every last green thing on the planet. But they don't."

The "top down" explanation, as Schultz puts it, is that the insects themselves are eaten so fast that there are never enough left to consume all the plants. The "bottom up" possibility is that plants' defenses work well enough to thwart the insects. Most likely, both of these are true to some extent. But here's another puzzle: If plants are producing chemicals that work as poisons, and if they do it year after year, "it would be the same as applying a pesticide," reasons Schultz, "and you'd expect the insects to evolve around it, but they don't do that. We don't have that problem in nature. Even those insects well-adapted to various plants rarely defoliate them."

Scientists know that plants respond actively to their environment. Plants form symbiotic relationships with beneficial fungi, acclimate themselves to low temperatures, detoxify air pollutants or tolerate them, manufacture antimicrobial chemicals, and brace themselves against a steady wind. But how these mechanisms work cannot be explained within any one field of biology.

To investigate such puzzles, Schultz and plant pathologist Eva Pell have created a research training program structured so that graduate students will make conscious links among three tiers of inquiry: molecular and biochemical mechanisms, physiological (or "whole plant") responses, and ecological consequences. Twenty-four scientists from agronomy, biochemistry and molecular biology, chemical engineering, entomology, forest resources, horticulture, plant pathology, and veterinary science have joined Pell and Schultz under a $1.3 million National Science Foundation grant to set up a five-year program of courses, discussion groups, and faculty collaboration.

In September, after "an intensive and very competitive recruiting campaign," says Schultz, six of the "top plant-science graduate students in the nation" came to Penn State. "They're now knee-deep in their first year of study," he says, "getting course requirements out of the way and starting their first lab rotations" — short-term research projects in the labs of the various faculty members. "They're cloning genes from maize, switching on plant defenses with airborne signals, visualizing membrane activity during root growth with jellyfish proteins that glow, studying the behavioral responses of roots to varying soil conditions, using lasers to measure the electrical conductivity of plant cell membranes, and heading off to the jungle to watch plants respond to leafcutter ants." Next summer they'll work as a team, in what Schultz calls "a sort of intellectual Outward Bound program," to solve an assigned problem in ecology.

"We're not trying to train a student to have the equivalent of a full Ph.D. in molecular biology, physiology, and ecology all at the same time," Schultz explains. "There aren't enough years in a career to do that. Students will receive the standard disciplinary training of whatever department they adopt as their 'home base,' with the same rigor as any other Ph.D. student. But they'll also learn how to talk to people not working in the same field."

He shrugs. "I was trained by an ornithologist to use a notebook and a pair of binoculars. Today, if I don't know what the various molecular methods are, I can't do ecology anymore."

# On a bright cold spring morning I follow Pell in quick-step from her office to one of a dozen greenhouses lined up like sparkling jewelry boxes behind Eisenhower Auditorium. She walks with the determination of one who knows by habit and necessity the absolutely shortest distance between two points.

We step through the wooden doorway and the familiar warm close breath of plants envelopes us. I'm a little startled to see so much motion: Four "continuous stirred tank reactors" are lined up, head-high and a good four feet in diameter, each with eight potted plants inside experiencing the equivalent of a personal hurricane.

"We want to get maximum uptake of ozone in these plants," Pell explains; a plant pathologist, she is investigating the effects of ozone. "We need the constant agitation to make sure the plant is fully exposed." A fan on the top of each closed tank keeps up the eternal wind. She points to a sizable vent on the wall. "All the air that comes in here passes through the charcoal filters, which remove any pollutants. Each of the tanks has a large intake pipe for filtered air and a tube through which we can introduce a specified amount of ozone. The outlet pipe leads to our command center, where we measure the levels of ozone quite precisely. Would you like to see the command center?"

Crammed into a closet just big enough are a chair, a table, a computer, and tanks of ozone. Via tubes, the computer measures and delivers the desired levels of ozone to each tank, and monitors the actual levels achieved. Half the plants are receiving about four times the ozone typically found in outside air; the other half are controls: their charcoal-filtered air has almost no ozone.

Damaged by stresses like ozone, plants can lose biomass, lose energy-producing leaf area, and presumably become less "fit" for reproduction. Pell is also interested in how a plant compensates for this stress. Besides turning on production of defensive chemicals, stress can cause a plant to shift its "carbon allocation" to emphasize growth in areas where it will do the most good. Suffering drought, for instance, many plants shift carbon to their roots. When exposed to photosynthetic poisons like ozone, plants will favor the shoot: older leaves will age prematurely and drop. This "accelerated senescence," Pell thinks, could be a coping mechanism. But unraveling the biochemistry to see if it actually helps the plant proves to be a tricky business.

The current experiment focuses on the effects of ozone on a plentiful plant protein, Rubisco, in tobacco. An essential enzyme in photosynthesis, Rubisco takes up CO2 and binds it to a sugar skeleton to begin the cycle that produces sugars and starches. Rubisco is manufactured and broken down constantly. Fifty to 70 percent of the soluble protein in a leaf consists of Rubisco, but as a leaf ages the amount gradually declines. Interestingly, ozone exposure also appears to reduce Rubisco levels, leading Pell to speculate that the loss of Rubisco may help to accelerate leaf drop.

Genetic engineering techniques have made studies of such biochemical pathways easier, Pell notes. Work by Barbara Zilinskas, one of Pell's collaborators at Rutgers University, has produced a transformed tobacco plant with a gene that may protect plants from ozone toxicity, Pell says. She and her colleagues at Penn State, meanwhile, are developing transformed potato plants that are missing a gene for an enzyme thought to regulate the synthesis of ethylene, a hormone high in ozone-stressed plants. "The beauty of using transformed plants," Pell says of this new technique, "is that we can target one specific protein and see the effects of its absence. Chemical inhibitors often are not specific enough to provide that accuracy."

Pell could have missed the fact that mildly ozone-stressed plants also develop resistance to diseases, she notes, if she had not "always enjoyed thinking broadly." Collaboration is an "essential" part of her personality. "To me," she emphasizes, "the fascinating questions are at the interfaces between disciplines."

# Schultz leans forward over the seminar table. "I like to know why the world is the way it is. How it works — why this way, as opposed to some other way."

I imagine what Schultz might have been like as a boy: collecting thousands of insects in jars, tearing apart flowers, uprooting bushes to examine root hairs . . . Nothing seems to excite him as much as a good natural "mystery."

He notes a recent study, for instance, in which a researcher found that the "bumping" of a bee as it flies into a flower to collect pollen will stimulate the plant to synthesize a group of chemicals that previously have been regarded as defensive. If humans worked the same way, loosely speaking, we might produce antibodies in response to a kiss.

"Almost anything you can think of that comes in contact with a plant, chemically or physically, appears to switch on at least some of the same mechanisms that are switched on in response to 'damage.' We don't quite know why that is," Schultz says, "but here something happy is happening to the plant, and what seems to be a defense gets switched on."

Moment to moment, plants allocate their energy and nutrient resources to serve the best interests of the organism. Growth and reproduction are generally a priority. But plants can invest a good deal in what's called "secondary metabolism": the manufacture of compounds that don't play a primary role in the life of the plant. Many of these chemicals seem to have little or no activity in the plant — but are very active in other organisms. Nicotine and caffeine, for example, are strongly active in nerve cells, which plants don't have; by hyperstimulating nerve transmission, they prove to be potent insecticides (as well as stimulants for humans).

Some of a plant's secondary metabolism goes on all the time, making such defenses as sticky hairs that trap insects, sticky sap like the latex in a rubber plant, thick coatings on leaves that reduce moisture loss, and lignin and cellulose that toughen the stem against bites while holding up the plant. The defensive array also includes poisons. Pine-sol, the household disinfectant, contains monoterpenes common in conifers and deadly to fungi, bacteria, and viruses. Clover, innocent-looking to us, contains cyanogenic glucoside, a sugar with cyanide bound into it. When an insect eats clover, its own enzymes break down the sugar, releasing enough cyanide to kill it.

Induced responses occur after the plant perceives something in its environment. Cold temperatures, changes in orientation, physical contact (by the wind, direct damage, touch, or rubbing), and chemical contact (such as the presence of a toxin, fungus, virus, insect, or bacterium) all can stimulate chemical changes in a plant. One clever and fairly common induced response is called the hypersensitive response: the cells closest to invading microbes simply die, sacrificing themselves to form a "wall" around the invaders and effectively starving them out, since viruses and bacteria need living cells to survive. (This response is visible as spotting on the leaf surface.) Of course, plants respond to good things too. They will form special structures in their roots to provide a place for helpful bacteria, or selectively allow certain fungi to invade root cells.

The message that a stimulus is present is thought to be conveyed through the plant by "signal" molecules. In this way, one leaf experiencing a threat (or the presence of something positive) seems to communicate to the rest of the plant what is happening to it, and the whole plant responds.

"Sometimes there are multiple signals, or several steps between the injury and the gene expression," says Schultz. "The molecular biologists have approached this phenomenon from the gene level and are working their way towards the products, while chemical ecologists like me have approached it from the products and are working our way back toward the genes. We're hoping to meet in the middle!

"Rather few molecules," he continues, "maybe eight or ten, have been identified as signal molecules in most plants studied, yet there are lots of kinds of plants and lots of different responses. Salicylic acid — basically, aspirin — turns up in almost every response to a microbe, whether good or bad, as well as in responses to cold or drought. Jasmonic acid is involved in most responses to physical stress or damage. Ethylene functions to coordinate flower maturation and fruit ripening, and in many plants also appears to be an essential step in developing resistance to a pathogen. For each one of these few molecules we can identify a role it has in a variety of different functions."

Yet a plant can often recognize a particular species and even genotype of fungus, distinguishing a beneficial or benign type from one that will cause damage. "How does a plant tell those stimuli apart?" asks Schultz. "How does it come up with different responses? And how does it turn that into a distinctive response that deals with that particular stimulus?

"Or does it?" Schultz is not one to leave any stone unturned.

# Pell and Schultz had not collaborated previously when they discovered each was thinking along the same lines.

"After one meeting," says Pell, "it was obvious that it would be better to join forces than to compete for the grant. And it was really exciting — although we are very different as people, we had come to some of the same ideas."

The first of these ideas became the basic hypothesis behind the group's research: that plants are more similar than we might think. Most members of the group expect plant responses to be integrated by a limited number of molecular and physiological mechanisms. Identifying and understanding these should provide insights to a range of other problems, "from designing better plants to understanding the evolution of species," according to Schultz. The goal is to make the plant sciences predictive, with principles that allow scientists to anticipate environmental effects, not simply try to explain them once they happen.

The second idea that brought Pell and Schultz together was that graduate training should require collaboration. To Pell, "it's a matter of the mind set. The molecular biologists work on fundamental questions and they get caught up in that world, rarely asking, 'What are the implications for the plant in the environment?' Ecologists do tremendous studies looking at the behavior of populations or at how a plant performs, but have rarely tackled the question, 'What's the mechanism by which the plant responds this way?' In the traditional educational pathways, there's just not the desire or the intellectual preparation to look at the interactions among different levels."

But, Pell adds, "We're in a time of transition in science. The 'star' system is changing. We need each other more than we used to, because technology is giving us opportunities to do things we just can't do by ourselves."

Notes Schultz, "We're not necessarily going to resurrect the true generalist scientist here, who has Ph.D. level expertise of enormous range. Instead, we'll retain the power that comes from an in-depth focus, but build teams of those powerful people."

In the final phase of the elaborate NSF grant review process, after a full day of presenting their ideas with next-to-no feedback, Pell was pleased to hear one NSF representative say, "In other words, you're going to educate biologists. That's something that hasn't been done for 20 years." And Pell thought to herself, "Bingo."

Eva Pell, Ph.D., is the Steimer Professor of Agricultural Sciences in the College of Agricultural Sciences, 321 Buckhout Lab, University Park, PA 16802; 814-865-0323. John C. Schultz, Ph.D., is professor of entomology in the College of Agricultural Sciences, 103 Pesticide Lab; 863-4438. This project is funded by the National Science Foundation.

Last Updated March 01, 1996