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Taking apart and rebuilding plant genes.

Plants constantly struggle as they sprout from seed, grow, mature, and bear fruit and new seed. But no sign of this struggle is visible when a friend hands you the perfect home-grown tomato or you drive past the unblemished green of a young soybean field.

That's because these delights are tricks of nature. But how, if not by magic, do plants assemble a tomato or a soybean seed?

They do it through the biochemical traffic in and out of the cells, the plant's living engines, piloted by genes.

Genes direct cells to build proteins, hormones, toxins, and other chemicals. With these raw materials, cells create and ship energy, get rid of waste and invaders, and grow the cell and tissue structures the plant needs to survive, grow, and mature.

Today, scientists at the Agricultural Research Service are making plants reveal more of their genetic instructions. Still, only a tiny fraction of genes have been inventoried.

At the Plant Molecular Biology Laboratory (PMBL) in Beltsville, Maryland, the scientists are also redesigning genes: in soybeans, tomatoes, peaches, rice, potatoes, sugar beets, and other crops.

The eventual payoffs could be healthier plants, more economical farming, and more nutritious food on the table.

The red brick laboratory building is a plain-looking vessel for the scientific enterprises simmering inside, with 10 full-time ARS researchers along with support scientists, research associates, technicians, and collaborators from both the University of Maryland a few miles away--and faraway lands such as Israel and South Korea.

Genetic Cartography

PMBL geneticist Thomas Devine and several colleagues are looking very closely at genes in the soybean plant.

An $11 billion crop in the United States in 1991, this plant supplies two-thirds of our oil for food uses. The oil also has dozens of industrial uses, and the seed's high-protein meal sustains people and livestock around the world. So the job is not to put the soybean on the map. Instead, Devine is helping put a genetic map on the soybean. To visualize a gene map and how genes act, it helps to know a little about railroads and, as you' 11 see, music and poetry.

On a map of a rail line, you can locate stations along the way and find the distance between them. But in a cell's nucleus, the stations--the genes--are bunched up next to each other along molecular rail lines called chromosomes.

Imagine two giants picking up either end of a railroad line and twisting the tracks in opposite directions--with the rails staying spiked to the railroad ties.

That gives you a fair image of the double helix--the molecular structure of chromosomal DNA, or deoxyribonucleic acid. Scientists discovered DNA's structure 30 years ago, although they began unmasking its logic of inheritance decades earlier.

Linkage: The Bond That Ties Inheritance

You can mark rail distance in miles, and Devine and other scientists can measure nucleic acids by counting their components--individual nucleotides.

But, he explains, scientists map the proximity of two genes by what really counts: the likelihood of both being passed on together to offspring. In soybeans, for example, a black seed coat is commonly inherited in tandem with resistance to nematodes.

The higher the probability of two traits being inherited, the closer the linkage of their genes. Closely linked genes are near each other on a chromosome, and scientists use a crucial bit of logic to expand the known network of genetic linkage. "If one gene is closely linked to each of two other genes, those two are linked to each other," Devine says.

Gene linkage makes for more practical breeding strategies. With a complete gene map, Devine says, "a breeder could estimate how many progeny would be needed to grow from parent plants to get, say, 50 test plants with a desired combination of genes."

His efforts are part of an extensive USDA research program on plant genome mapping, coordinated by the Agricultural Research Service. More than 300 ARS and university scientists around the country are participating. Devine collaborates on soybean mapping with Beltsville colleagues and scientists at two other ARS locations and six universities.

Two gene maps hang on a wall in Devine's office. One is a rudimentary classical map of 19 linkage groups developed over the decades by plant breeders and geneticists. The other map, still evolving, integrates classical linkages with those being found through new molecular approaches.

With classical methods, it took Devine 8 years to map a gene that makes soybean stems grow in a flattened, almost inside-out manner called fasciation. "Now," he says, "we can use molecular probes to look at hundreds of points along the chromosomes and examine tens of thousands of linkage possibilities. This makes gene mapping 4 to 5 times faster."

A molecular probe is a specific--usually unknown--sequence of nucleotides. With a variety of tests, scientists can often find whether and where all or part of a sequence occurs in a plant's chromosomes.

"integrating the classical and molecular maps will yield a comprehensive map of agronomically important genes. The molecular approach is a bridge to that map," Devine says.

On his molecular map, linked genes have cryptic names--like Rhg4, BLT24, and BLT65. Someday, the linkage of these genes by the lab's researchers could--for starters--help U.S. soybean growers overcome a microscopic, wormlike pest that carries a $250 million annual price tag.

Breeders have known that the Rhg4 gene enables the plant to resist the root-damaging soybean cyst nematode. But Devine and PMBL colleagues found that this gene is closely straddled by the other two--BLT24 and BLT65.

"This discovery," Devine says, "could lead to isolating the Rhg4 gene and transferring it into susceptible plants and varieties, making them resistant to nematode infestation."

Other scientists at the lab are finding that the two straddling genes could also have payoffs of their own. Eliot Herman found that the BLT24 gene encodes a protein made only in cells of developing seeds.

Benjamin Matthews, who cloned the other straddler, BLT65, says it makes an enzyme, aspartokinase-homoserine dehydrogenase, important in producing the amino acids lysine, threonine, and methionine. Matthews, an ARS plant biochemist, adds that "Cloning this gene could give us a new strategy for improving nutrition, not only in soybeans but also in other crop plants."

Using molecular probes, Matthews, Devine, and other researchers--Gordon Lark of the University of Utah and Reid Palmer of ARS in Ames, Iowa--have added about 200 new gene "stations" to soybean's molecular map, including 25 new linkage groups. They also found "station addresses" on soybean chromosomes for traits tied to the plant's height, leaf area, seed oil, maturity, and susceptibility to lodging.

"Once we find a gene's linkages--particularly with genes we know are tied to important traits," Matthews says, "we can home in on the genes that breeders and genetic engineers may want to exploit to improve a plant."

Fueling the Amino-Acid Machines

"We don't yet know enough," Matthews says, "about how cells perform some functions"--such as how leaf chloroplasts use carbon and nitrogen to build amino acids they ship out to nourish and fatten seeds.

In 1991, Matthews and former research associate Gregory Wadsworth found that soybean seedlings make five different forms of an enzyme, aspartate aminotransferase (AAT). AAT helps the plant control its balance of carbon and nitrogen, but "each form of ATT may have a unique role," Matthews says.

Recently, the scientists cloned a gene that produces one of the AAT enzymes. When they inserted the gene into bacteria, the microbes obediently cranked out the chloroplast form of AAT.

"This not only is a way to produce large quantities of the enzyme," Matthews says. "It also lets us alter the sequence of the AAT gene and use the bacteria to produce an altered and more efficient enzyme and, ultimately, to boost the soybean's nitrogen use and yield."

Matthews is also working to raise soybean's nutritional value. For this he turned to the common carrot. "It's relatively easy to put new genes into carrot cells and regenerate whole plants," he explains. "With soybeans, regeneration technology isn't quite there yet."

His aim is to raise soybeans' production of methionine, an amino acid soybeans currently make little of. More methionine would give soy meal higher nutrient value for animal feeds--and for foods people eat.

First, Matthews and colleagues isolated a carrot protein that acts to make two different enzymes during its synthesis of several amino acids. Later, they isolated and cloned the gene governing this dual-purpose protein.

Matthews and former PMBL research associate Jane Weisemann are patenting the gene and its use. "We want to see if we can alter this carrot gene to regulate the amino acid content in plants--in particular, lysine, homoserine, threonine, isoleucine, or methionine," he says. That could lead to better balance of amino acids in crops: more methionine in soybeans and more lysine in rice, for example.

Gene-Fest for Tomorrow's Champion Crops

Gene mappers aren't interested only in soybeans--or only in plant genes, for that matter. In searching for ways to give crop plants new genes to ward off disease, PMBL scientists are also examining genes from fungi, moths, and viruses.

Peter Ueng, a plant pathologist, wants to give wheat a better chance of surviving Septoria fungi. As the fungi invade and destroy cells, they mar leaves and outer seed husks (glumes) with ugly brown blotches.

More common in the humid Southeast, leaf and glume blotch can cause a 10- to 40-percent drop in yield, says Ueng.

But on some wheat leaves, the blotches have yellow halos, a sign that the plant's cells are putting up a molecular struggle to halt or slow the fungus. Now, Ueng and university colleagues have narrowed down which genes enable cells in the haloed areas to fight back.

"Our ultimate goal is to reduce yield loss and the farmer's need for fungi-cides by giving wheat new, powerful genes to stop the disease," Ueng says.

But the first big advance, he adds, will be an ability to forecast the virulence of new strains of Septoria that regularly show up. Ueng found that the fungus has an unusually large natural genetic variation. By comparing the virulence genes of known and emerging strains, he says, scientists could give farmers an early alert.

Ueng's co-researchers are with Cornell University and Purdue University; such collaboration is the PMBL's hallmark. Since January 1991, the lab's scientists reported research findings with 75 scientists at 18 ARS labs, 20 U.S. universities, and institutions in Belgium, Canada, Egypt, France, India, Israel, Italy, Qatar, South Korea, and Yugoslavia.

Down the stairs from Ueng's office, plant physiologist Lowell Owens directs and collaborates on several studies to give novel genetic defenses against bacterial wilts and rots to sugar-beets and other crops. His cooperators include former research associate Russell Nordeen and scientists from Beltsville's Livestock Insects and Vegetable laboratories, the University of Alaska, and Louisiana State University.

Moth Genes Fly Bacterial Relief Missions

Pseudomonas bacteria are among Owens' chief targets. Infection starts when a root, shoving its way through soil, scrapes itself as it creeps past a sharp particle of sand. That can allow bacteria to invade and set up shop in the plant's xylem tubes, which can3, water to stems and leaves. As the bacteria multiply, they make a gum that clogs the tubes. Leaves--and soon the plant--wilt and die.

In recent years, scientists looking for natural antibiotics have been testing genes from not only plants but also fungi, bacteria, and moths. A gene in the giant silk moth, for example, enables the insect to make cecropin, a small protein that fights bacterial infection. Owens has inserted a modified cecropin gene into tobacco and is testing to see if it protects the plants from wilt caused by Pseudomonas solanocearum bacteria. [See box on page 8.]

Earlier, Owens collaborated with Steven Sinden of the Beltsville Vegetable Laboratory to test the feasibility of protecting potatoes from soft rot with a cecropin gene or a gene from chickens that makes an antibiotic known as lysozyme. In tests so far, lysozyme appears to have the edge.

Potatoes with cecropin and lysozyme genes have been tested in outdoor trials coordinated by ARS scientist William Belknap in Albany, California. Some results look promising, but they're far from conclusive.

An antibiotic could be worse than a disease if a plant got too high a dose, says Owens. But he, Sinden, and Nordeen found that cecropin is toxic to nine bacteria at levels far below those at which it harms protoplast cells of soybeans, sugar beets, and sweetpotatoes. However, tomatoes and some potato varieties are more sensitive, so making cecropin work without harming these plants may be difficult.

Harmonizing With Hormones

To know if a new gene can benefit plants, PMBL scientists first have to make cells reveal more about how their existing genes run the business. To harmonize the plant's chores of producing and storing its food and growing to maturity, cells generate a key hormone called cytokinin, says geneticist Ann Smigocki.

"Cytokinin has a broad role in the normal growth and development of a plant. It carries out this role partly by controlling the expression of many plant genes," says Smigocki.

To make cytokinin reveal its genetic tinkering, she has genetically engineered several plants including tobacco, tomato, peach, and others. She turns cytokinin on and off by equipping these plants with a different cytokinin gene--one whose DNA switch, or promoter, she can control.

Some promoters originate from genes in plants; others, from insect genes. One promoter--from fruit flies--turns on one of the genes that help this insect avoid or recover from heat shock.

Smigocki hooked this promoter to a cytokinin gene and engineered the assembly into tobacco. After exposing plants to 125[degrees]F heat for an hour, she found much higher levels of several proteins. The heat-activated promoter had roused the cells to make cytokinin. Using this and other gene switches, Smigocki and research associate Scott Harding have isolated more than 200 cytokinin-affected genes, and are closely examining about 70. They want to match up the genes to the proteins whose levels rise or fall when cells flank out cytokinin.

Smigocki says identifying the proteins--and how, when, and where they act--will help reveal how to manipulate their associated genes for better yield or other desirable traits.

One promoter/cytokinin assembly she uses to track the hormone's influence may enable plants to act quickly to retard caterpillars and other attacking insects. "What we've done is get plants to overproduce cytokinin when they're being eaten," says Smigocki, who is patenting the approach. In lab and greenhouse tests, tomato hornworms ate and grew at less than half the normal rates when their diet was leaves from plants harboring the new gene.

When Cells Lose Their Grip on Light

As a growing plant struggles to cope with insects and diseases, its leaves must continue harvesting light to make food--the business of photosynthesis.

Why, then, will a perfectly healthy bean leaf turn yellow and drop off during the course of a few days? This apparently trivial event has huge implications, says plant physiologist Autar Mattoo, who is head of the PMBL.

When a leaf matures, he notes, it simultaneously stops growing and loses the ability to capture sunlight and convert the atmosphere's carbon dioxide into food. He and colleagues--Roshni Mehta of the University of Maryland and former research associate Timothy Fawcett--uncovered some of the gene-driven events responsible.

"This is taking us closer," Mattoo says, "to genetically engineering crops that can make their own food for a longer, or shorter, time during the growing season. That could lead to earlier harvests or later, larger harvests and more nutritious crops."

In studies with wheat and an aquatic plant, duckweed, the scientists used copper ions to artificially age leaves in a few days. That made it possible to measure biochemical changes in the chloroplasts--the hundreds of gene-filled foodmaking factories in each leaf cell.

Mattoo and colleagues found that the aging process yielded hordes of a type of oxygen atoms known as free radicals. These atoms-run-amok have been associated with a breakdown in growth processes and disease defenses in humans, livestock, and other animals.

In Mattoo's test plants, the free radicals rearranged an amino acid in rubisco, a photosynthetic protein.

How important is rubisco?--By changing carbon to a form plants can use, it makes life possible on this planet. But retooling the amino acid in the test plants inactivated their rubisco.

"Dead" rubisco proteins travel to the chloroplast's membrane and are then disassembled by enzymes, Mattoo explains. Some rubisco elements--nitrogen, for example--are sent to other parts of the plant, such as seeds that are filling or leaves still on the light-grabbing side of maturity.

"The plant recycles what it can use and sends any surplus to the soil, via its roots," Mattoo says. "We still need to identify the genes and the mechanisms controlling this and other processes that shut down the chloroplast factories. Then we can see how photosynthesis is affected in plants given a variety of differently reconstructed genes."

Reducing Solar Energy Waste--and Sunburn

The shutdown of chloroplast factories isn't Mattoo's only concern. Plants have had billions of years to perfect their photosynthesis machines, but they seem to vastly underuse their energy resources.

"Plants can use only about 2 percent of the sunlight reaching their leaves," he says. And he's fingered D1--the key protein in the chloroplast's membrane-as one of the culprits. "Plants expend enormous energy," he says, "to rapidly make and then degrade this protein in sunlight--more rapidly if given extra ultraviolet light. We want to know how and why."

Earlier, the scientists found that some weed-killing chemicals bind to D1, block the movement of electrons through it, and inhibit its breakdown. Now they've found that these are three separate, not linked, properties of the D1 protein. "We are looking into this further," he says, "because we want to use bioengineering to slow D1's degradation and then analyze the plants' photosynthetic efficiency."

Last year, Mattoo and Marvin Edelman and others at Israel's Weizmann Institute of Science discovered that ultraviolet-B radiation destroys the D1 protein.

UV-B is a part of the light spectrum known to cause skin cancers in some people who get too much sun over a prolonged period of time. Much of the sun's UV-B is screened by Earth's natural ozone layer, but pollution has thinned the ozone by 5 to 10 percent in the past 20 years.

Mattoo says the new finding about D1 lends new significance to ozone depletion. "An increase in UV-B reaching Earth could eventually translate into less crop productivity--and less food. That's because UV-B, by destroying D1, reduces a chloroplast's efficiency in carrying out photosynthesis," he says.

He and former research associate Tedd Elich also found a change in D1 under normal light. "A phosphate molecule lodges onto one of its amino acid building blocks," Mattoo says. "This may have to happen before light can break down the protein."

The next step is to see how minor alterations to D1's gene affect a plant. "That will gradually tell us what turns this gene on and off. In other words, what tells the gene when to build and when to destroy D1 protein," Mattoo says.

This approach is sometimes tagged with the violent-sounding title of "gene bashing." But it can be compared to the editing of poetry. The verses are linked fragments of genes; the syllables are nucleotide sequences. Editing is done with probes--biochemical "blue pencils" that scientists use to delete one to thousands of nucleic acid syllables.

If editing quickens or slows the biochemical rhythms under study, scientists know they've touched--or bashed--a gene they want to know better.

Dying on the Vine: It's Natural

Remember that falling bean leaf?. While Mattoo wants it to have a longer, more productive youth, molecular biologist Mark Tucker wants to know why it detached itself, or abscised.

Tucker also wants to know why flowers sometimes take a dive instead of staying on to produce fruit, and how to get ready-to-harvest crops such as apples, beans, cotton, oranges, nuts, and tomatoes to cooperate better with hand or machine harvesting.

Using a scanning electron microscope, Tucker and colleagues examined the area of cells on the abscission layer, where stem and leaf perform the delicate biochemical surgery.

Abscission does not rupture the cells, they found. Instead, the cells themselves dissolve the "glue" that had bound a single layer of stem cells to cells of the departing leaf.

Stem cells, says Tucker, team up to amputate a leaf with little or no "bleeding" of the stem's moisture or nutrients. The cells wield an enzyme, cellulase, for this surgery.

"It's been known for some 40 years that cells make cellulase and that it breaks down some component in the cell's wall," he says. "We need to find out more about how genes and hormones interact to drive this process, so we can modify the genes to control it."

Scientists already know the key hormones: Ethylene promotes abscission, and auxin retards it.

Earlier, Tucker and collaborators identified and sequenced a cellulase gene from beans. When the gene springs into action, it makes messenger ribonucleic acid, or mRNA. This mRNA acts as the cell's work order: "Make cellulase now!"

In abscised leaves, Tucker and colleagues found that cellulase mRNA occurs only in the two layers of cells on either side of the separation zone and in the vascular tissue that feeds the leaf.

Currently, he's examining tomato plants to which he gave different sets of the cellulase gene from the bean plant. The strategy is to edit the cellulase gene until it reveals its promoter, or switch.

"We would use the promoter," he says, to make a substance such as auxin. That would stop the gene from making cellulase when we don't want it made-- for example, when it causes premature abscission in flowers and fruit."

He works with tomatoes because the cells are amenable to gene engineering and regrowth into whole plants. But one of his aims is to reduce flower abscission in soybeans.

Like most plants, soybeans make more flowers than necessary--as insurance. "About 70 to 80 percent of soybean flowers drop off prematurely," he says, "because of drought, insects, or other stresses. But you wouldn't want all the flowers to stay on anyway, because the plant couldn't support all the resulting seeds."

Other scientists plan to control abscission by exploiting the front end of the biochemical chain of events.

The strategy is to alter a soybean gene for an enzyme called ACC synthase. This enzyme is the key controller over the hormone ethylene, says Mattoo. Ethylene stimulates cells in the abscission zone to make cellulase.

"We cloned this soybean gene and inserted it into bacteria, which then produced the enzyme," says Mattoo, who did the work with PMBL research associate Ning Li and University of Maryland graduate student Derong Liu. "Eventually, we want to genetically engineer soybean plants to shut down ACC synthase. That would stop ethylene, cellulase, and as a consequence, abscission."

Cell Suitcases Stuffed With Oil, Protein

Soybean plants may be too free with their flowers. But while this means fewer seeds for growers to harvest, it's part of the plant's reproductive strategy. To carry out the strategy, chloroplasts in leaves prepare and ship the ingredients, but what do seed cells do with them?

By finding answers, PMBL scientists hope to engineer a soybean to serve the grower's purpose, not just its own.

That means they must--like auditors-make seed cells answer for the resources they accumulate and spend.

Plant physiologist Eliot Herman and colleagues delve into the seed cell to find out how its genes control machinery for churning out oil and proteins and storing these for later use when the planted seed germinates.

In cells of a developing seed, Herman explains, four key proteins form molecules of oil into thousands of microscopic droplets. Each droplet is wrapped in a membrane made of these proteins and phosphorus-rich lipids. No membrane means no droplet.

Herman and research associates Deborah Loer, Andy Kalinski, and Daniel Rowley are taking apart genes that tell cells to make the membrane proteins. "We are trying to find out how these genes are turned on and off in response, perhaps, to environmental stresses such as drought, heat, and inadequate nitrogen,"he says.

The scientists identified and cloned two genes responsible for the most common membrane protein, 24 kDa oleosin. "We're using the clones," Herman says, "to find out how the cell controls the accumulation of oil bodies. We may be able to alter them to force the seed cells to make less oil and more protein for meal."

That protein is packaged in cell vacuoles, or large fluid-filled reaction chambers. They take in, process, store, and digest nutrients and expel waste and excess water.

Typical cells have one vacuole. Enclosed by a membrane, it occupies most of the volume of the cell. But in seed cells, the vacuole divides into thousands of small, protein-filled units. These and the oil droplets turn the cell into a stuffed suitcase of nutrients.

Having many small vacuoles means a vastly larger area of vacuole membrane, so a germinating seed can get at lots of protein in a hurry. "Subdividing the vacuole is the seed's way of chewing its food so it will be easier to digest later, when the seed sprouts," Herman says.

To digest stored protein, germinating seeds make yet another type of protein, thiol protease. Herman and Kalinski found that one thiol protease, P34, is made only in cells of still-developing seeds. They are now investigating whether it has a role in seed/cell protein storage.

"With the P34, oleosin, or other genes, we may be able to genetically alter the soybean plant to produce new varieties that yield less oil and more meal--or more oil and less meal. But to do that," Herman cautions, "we may have to get around a plant's habit of breaking down 'foreign' proteins. If these protein molecules are, for example, wrongly folded, the protein won't accumulate where we want it to--such as in vacuoles or oil-droplet membranes."

Folds in a protein molecule allow it to hook up to other proteins in a usable, three-dimensional manner.

To detect bad folds, cells make a special protein. Called binding protein, or BIP, it sidles up to wrongly folded proteins, perhaps triggering their breakdown. To learn how BIP works, Herman, Kalinski, and Rowley examine clones of BIP genes that govern folding of an altered storage protein from the common bean.

Why do this? The soybean industry wants to lift the crop's low sulfur content-to increase its value, especially as livestock feed. "This might be possible," Herman says, "by engineering the plants to incorporate more sulfur-containing amino acid blocks---cysteine and methionine--into proteins. But some of the bioengineered proteins are unstable. Therefore, to make it work, the folding problem will probably have to be solved."

Well-Rounded Meals--From Mutant Rice

Rice plants, like soybeans and other plants, pack a lot of protein into the seed--a high-energy jolt to start the next generation of plants. But Gideon Schaeffer's new rice--started from cells in the lab-could do a better job nourishing the next generation of people.

Last fall, he released the first five breeding lines of rice with protein that's high in lysine, an amino acid essential for human health. Commercial rice--like all major cereals--is low in lysine, and extra lysine would make rice a more nutritionally balanced food.

Schaeffer's high-lysine rice has slightly more total protein and 15 percent more lysine than parent plants from a commercial variety, Calrose 76.

"We first came up with high-lysine rice over a decade ago. Now we've stabilized the trait and defined some of the biochemistry so breeders can develop high-lysine commercial varieties," says Schaeffer, a plant physiologist. New varieties could be available to growers in a few years.

Rice is relatively minor in diets in developed countries, except Japan. But, he says, "high-lysine rice could improve the diet of people in developing countries, such as those in Africa and Asia where rice is the main--sometimes nearly the only--protein source." The most interest so far is from Chile, India, and China.

"Almost all rice," he adds, "is consumed in the same countries where it's grown." In 1990, only 3 percent--15 million metric tons--of the world's rice went on the international market. Two-thirds of the exports came from three countries: Thailand, Vietnam, and the United States, with the last accounting for about 3 million tons.

"Traditionally," he points out, "breeders in this country have been most interested in yield--that's the producers' first requirement. But high-lysine grain, with yield and other qualities similar to commercial varieties, should be worth more to grow."

To develop his high-lysine rice, Schaeffer used tissue culture techniques to select rice cells with a genetic mutation that kept them making lysine far longer. He did this by challenging rice embryo cells with high doses of lysine and similar compounds, which ordinarily would kill them. From surviving mutant cells he grew whole plants.

High-lysine seed looks chalkier--more opaque--than regular rice, which is quite translucent. Since past breeding didn't favor chalkiness, it may also have lowered lysine content, Schaeffer notes.

"Continuing studies of rice cells in tissue culture," he says, "give us an efficient and powerful system to examine how cells make amino acids and synthesize and transport protein."

To make a protein molecule, a seed cell has to assemble a jigsaw puzzle. Each piece is a series of amino acids whose order is fixed by the cell's genetic machinery. Genes also command the folding of each amino acid series into the correct shape for assembling proteins to be used in the cell or stored elsewhere.

Schaeffer's rice plants work longer at making lysine-rich proteins. He is identifying the messenger RNA's that are active when cells do this and is getting very close to identifying the altered genes. "When we can do that," he says, "we will try genetically engineering rice and other crops for higher lysine."

High-lysine cells in culture may have industrial applications. "Some of the high-lysine cell lines spit out twice as much protein into the cell growth medium, and the protein is more water soluble," Schaeffer notes. He says these traits could boost production of pharmaceuticals and other high-value substances.

Peach Orchards With a Cultured Pedigree

Cell culture has been carried almost to an art form by plant physiologist Freddi Hammerschlag. Her work shows how scientists are putting together all the pieces to build better plants.

Look in almost any peach orchard in the Southeast, she says, and you'll find a potential home for bacterial spot disease. Leaves and fruit of every peach variety are to some degree vulnerable.

The bacterium, Xanthomonas campestris, emits a toxin or toxins that attack cell membranes. If the cell can't put up a strong defense to neutralize the toxins, its membrane weakens. Bacteria invade and multiply; the cell dies.

For the last decade, Hammerschlag has been developing peach trees that put up a stronger cellular defense. And it's clear she's an ardent peach lover, when she shows off her hundreds of peach trees--living in a refrigerator in the building's basement. There, covered glass dishes brim with tiny green trees smaller than alfalfa sprouts.

Hammerschlag grows such trees--and their full-size orchard-dwelling off-spring--from clumps of cells. "I develop technology, not new plant varieties," she says. "We want people to use the technology to make healthier peach trees and higher quality peaches."

That's what she has in mind for Redhaven and Sunhigh, two commercial varieties. "Sunhighs are the sweetest, juiciest peaches I've ever eaten," she says. "But many growers won't have anything to do with this variety because it's highly susceptible to bacterial spot. Redhaven is only moderately susceptible."

Hammerschlag improved the resistance in trees derived from both varieties by inducing changes in the genes in peach cells, then finding and testing potentially useful changes.

"Genetic variation is what we look for," Hammerschlag says, and she triggers even more by using various hormones and nutrients to culture cells. "We can also determine," she adds, "that only cells with a certain type of genetic variation will survive the tissue culture cycle." How? By adding to the culture medium the toxins produced by the bacterium to help it develop and spread disease.

The only cells that survive have a genetic ability to withstand the toxins. The approach, called in vitro selection, is also how Gideon Schaeffer developed high-lysine rice.

With cooperators in North Carolina, Hammerschlag now has the first evidence that her strategy works against bacterial spot in an orchard.

She's pursuing the same strategy with apples and another bacterial disease, crown rot. And she and cytokinin expert Ann Smigocki recently took tissue culture selection a step further. They accomplished a first by regenerating peach plants from embryos to which they had given a new gene for this hormone.

"Cells with the new gene produced many shoots," says Hammerschlag, "and the leaves had levels of two cytokinins that averaged over 50 times higher than leaves of unaltered plants." When grown to maturity, the trees were more compact--a potentially useful trait in high-density orchards, she notes.

Hammerschlag and Lowell Owens, meanwhile, are trying to transfer other foreign genes into peaches, including a moth gene for the cecropin protein. Can the moth protein control bacteria blamed for leaf spot and canker in peaches and fire blight in apples and pears?

Tests may reveal the answer to this question in a few years. By then, however, the lab's research team will be laying the gene railways of plants toward other new destinations.--By Jim De Quattro, ARS.

Scientists mentioned in this article are with the USDA-ARS Plant Molecular Biology Laboratory, Bldg. 006, Beltsville Agricultural Research Center-West, 10300 Baltimore Ave., Beltsville, MD 20705. Phone (301) 504-5103, fax number (301) 504-5320.

Peach Trees That Fight Back

In 1987, ARS scientist Freddi Hammerschlag and David F. Ritchie and Dennis J. Werner of North Carolina State University planted more than 150 peach trees in an outdoor test for resistance to bacterial spot.

Some of the trees at the university test site were conventional Sunhigh and Redhaven grafts on standard rootstock.

Others were propagated in tissue culture from axillary buds. This latter technique, called micropropagation, yields large numbers of plants that are clones, or genetic twins, of a parent plant.

About 100 of the test trees began as immature embryos five Sunhigh and two Redhaven--that Hammerschlag removed from seeds. She nurtured the embryos to form cell cultures and then small plants, and she cloned identical trees from each plant.

Several of the embryo-derived trees were more resistant to bacterial spot in the 1990 and 1991 field tests. The most resistant were grown from cells derived from a Redhaven embryo. Bacterial spot damaged only 13 percent of these peaches, compared with nearly half the peaches from Redhavens that hadn't undergone tissue culture.

Another set of trees came from toxin-resistant cells of a Sunhigh embryo. Fruit damage was considerable-but about one-third less than on standard Sunhighs.

Next, Hammerschlag will evaluate the offspring of these top-performing plants. "We got a lot of new information from those tests," she says. "For example, several genes apparently help the plant resist the disease. We'd like to isolate those genes so we can improve disease resistance even more."

Genetic Sheet Music for the Cellular Orchestra

When Lowell Owens describes the modified cecropin gene he inserted into plant cells, it's clearer why such work is called genetic "engineering."

Cells, after all, are engines: They capture energy in various forms and convert it into different forms. An orchestra is also an engine: Its players take in air and send out beautiful music, if they follow the musical score.

The genes of DNA are the cell's book of molecular composition. By making copies of itself called RNA, a gene prints the appropriate sheet music and the cell plays on cue.

Owens wants the cecropin gene's music to enable plant cells to kill bacterial cells--destroying their membranes so the bacteria can't multiply and clog a plant's water tubes or attack and destroy the plants' own cells.

The gene Owens works with has three sequences of nucleotides: a promoter to turn on the RNA copier, a coding sequence to make the bacteria-killing compound, and a third sequence to ensure that the compound goes outside the cell--where the bacteria are.

To work, the cecropin gene must first be turned on by a switch--a promoter. Gene engineers use promoters to borrow one organism's ability to respond to an event, so that another organism can respond--perhaps differently--to the same event.

Typically, Owens says, such an event is one or more of a huge number of specific conditions--such as heat or cold, wounding by insects or pathogens, low or high levels of sunlight, or the plant's developmental stage.

But unlike event-specific switches, the novel cecropin promoter "sings" almost nonstop. It's a piece of DNA from cauliflower mosaic virus. In the virus, its constant task is to multiply the virus particles. But for Owens' research, the promoter was snipped from that role.

Instead, its job is to nag the cecropin gene's coding sequence into continuously writing the nucleic-acid score the cell must follow to assemble choruses of cecropin.

The third sequence serves as the molecular finale. It modifies cecropin's biochemical tune so that, rather than hold on to the protein, the cell secretes it. Circulating through intercellular space around the plant's water tubes, cecropin can attack bacteria. If Owens and colleagues can get this strategy to play well in the lab--and then in greenhouse and outdoor tests--it could be welcome music to the ears of growers.

Anatomy of the Genetic Railroad

Chemically, genes are nucleic acids--the longest living molecules, made of linked units called nucleotides. A sugar, a phosphate, and a base--adenine, guanine, cytosine, or thymine--make up a single nucleotide. Along the genetic "rails," phosphates alternate with sugars. Each sugar is chemically "spiked" to a base. An adenine base at one end of each "railroad tie" always finds thymine at the other end. Similarly, guanine complements cytosine. A nucle-otide's base defines its characteristic identity, and the sequences of the four bases form each nucleic acid's unique structure.
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Author:De Quattro, Jim
Publication:Agricultural Research
Date:Jan 1, 1993
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