Arsenic Uptake, Transport and Accumulation in Plants

Project Leader: Mary Lou Guerinot Ph.D. Professor, Biological Sciences Dartmouth Medical School

Project Co-Leaders: Tracy Punshon Ph.D. Research Assistant Professor, Biological Sciences Dartmouth Medical School

David E. Salt Ph.D. Professor, Horticulture and Landscape Architecture Purdue University

Arsenic's lingering legacy

To fight the boll weevil in the 1950s, U.S. cotton-growers spread a pesticide that contained arsenic. An effective killer, the pesticide was also used against the gypsy moth. Yet it had an unexpected side effect: The soil of the fields and forests where it was applied remains laced with low levels of arsenic.

Arsenic can't be broken down to harmless by-products. It stays in the soil unless rain (or irrigation water) washes it into the groundwater, or a plant, like rice — which is now farmed on the old cotton fields — takes it up into its stems, leaves and grains. Neither of these outcomes is good for people, who might drink water or eat rice tainted with arsenic.

Number one on the U.S. Centers for Disease Control and Prevention's list of 50 chemicals of most concern for human health, arsenic has also entered farmers' fields by way of wood preservatives, leather-manufacturing waste, poultry litter, and the disposal of fly ash from coal-fired power plants. As part of Dartmouth's Toxic Metals Research Program, researchers Mary Lou Guerinot and Tracy Punshon are researching ways farmers and plant breeders can protect our food supply from arsenic and other toxic metals in the soil.

In large doses, arsenic is the infamous deadly poison. From the Roman Empire until 1836, when a test to detect its presence in a dead body was invented, it was the “King of Poisons”: a colorless, odorless, tasteless way of getting rid of an enemy by slipping a bit into a friendly glass of wine. The symptoms seemed like acute food poisoning. A dose the size of a pea brought death.

In the rural villages of Bangladesh, where shallow tube wells contain high (but not immediately poisonous) levels of arsenic from the natural rock formations surrounding them, long-term exposure has been linked to increased risk of cancer, heart disease, diabetes, skin cancers and reproductive disorders, among other diseases. Even at small doses long-term exposure to arsenic is dangerous. In 2001, the Environmental Protection Agency lowered the maximum allowance of arsenic in U.S. sources of public drinking water to 10 parts per billion.

From field to food

Some plants can draw arsenic and other metals into their leaves and stems at doses that would easily kill other plants. Scientists call these plants “hyperaccumulators.” Unfortunately, some food plants — particularly rice — are also very good at drawing in arsenic from the soil. In July 2008, scientists in Japan and the United Kingdom reported why: Rice mistakes arsenic (which does the plant no good) for silicon (which is essential)

Why would a plant make such a mistake? To a rice plant, says Punshon, arsenic looks like silicon because “these two chemicals have a similar shape and electrical charge.”

It's a common phenomenon: plants mistake toxic cadmium for essential iron, radiocesium for potassium, uranium for calcium. But in rice, the arsenic problem is magnified by how much silicon the plants take up — ten times as much as barley or oats. Silicon stiffens the rice stems and leaves, keeping the plants from falling over, or lodging, when their seed heads get heavy. It helps the rice plant grow stronger husks and also protects the seed against fungus. Rice breeders selecting for sturdy, fungus-free plants may have also bred for high-arsenic content grains without knowing it.

“And plants didn't evolve in the presence of arsenic,” adds Guerinot. Until the Industrial Revolution, most arsenic was tied up in rocks deep underground; through mining and the drilling of deep wells, we have brought it up to the Earth's surface.

The fact that rice grows best in flooded fields adds to the problem: flooding changes the chemistry of the soil dramatically and makes arsenic more soluble. At a Texas field site, Guerinot and Punshon's colleague Shannon Pinson of the U.S. Department of Agriculture grew 1600 varieties of rice under flooded and unflooded conditions. When their grains were tested for 18 different elements by colleague David Salt of Purdue University, Guerinot says, “We confirmed that rice grown in a flooded field picks up much more arsenic than rice grown in an unflooded field.” Chemically, the flooding causes iron compounds to precipitate onto the surface of the rice roots; the iron attracts arsenic, which binds to the roots and then is mistaken for silicon and transported throughout the plant.

Seeing where the arsenic goes

The most powerful influence on whether a particular type of rice takes up a great deal of arsenic or very little is the plant's genes. In that same study, the researchers found that strains of rice differ not only in the amounts of arsenic they take up, but how they distribute arsenic from the roots to the seed — the edible grain. The ideal rice plant for human consumption would be one that takes up very little arsenic, particularly in the rice grain that people eat. Guerinot and Punshon have chosen rice strains that are likely candidates for the ideal food plant, and they are working to block, or silence, the genes associated with arsenic uptake in those plants.

They are aided in this work by a high-tech way of seeing precisely where in the plant arsenic accumulates. The standard way of testing how much arsenic is in rice or any other material is to grind up a sample of grains, dissolve them completely and perform a chemical analysis. That method is not fine-tuned enough for Guerinot and Punshon. “That could tell us how much arsenic is in a particular strain of rice – but we want to know where the arsenic is.” Punshon explains. To determine that, she uses a technique called synchrotron x-ray fluorescence (SXRF) microspectroscopy to create detailed two- and three- dimensional images of the chemical make-up of the rice grain — rather like x-raying your bones to judge their level of calcium. With some adjustments, she hopes to be able eventually to image individual living plant cells.

Unlike other imaging techniques, she does not need to treat the rice grain with special chemicals, dry it, or cut it into thin sections — all of which change the structure of the plant cells and can add other elements which weren't there before. “We don't have to do anything to it but shine the x-rays on it.” When you shine a high-energy x-ray beam onto a seed — or any sample — its atoms become energized and give off x-rays of their own, like a fingerprint for each element. This is called the 'photoelectric effect' and is in fact the discovery that earned Einstein his Nobel Prize. The brightness of the fluorescence enables researchers not only to see how much of each elements is present, but where it is.”

The source of the x-rays Punshon uses is known as a synchrotron. “Basically it's a particle accelerator,” she says. “Synchrotrons are ring-shaped buildings in which electrons are accelerated to nearly the speed of light, steered in a circle by enormous magnets.” First built in the 1950s for physicists to study sub-atomic particles like quarks and bosons, synchrotrons emitted high-energy x-rays whenever the huge magnets steered the electrons, which although irksome to physicists studying the fundamental nature of matter, were incredibly useful for looking at what certain materials were made of. Now, synchrotrons are built specifically to harness this “leakage,” which is used in archaeology, forensics, geochemistry, and other disciplines to identify the make-up of materials. “The bigger the ring, the more brilliant the beam, and the finer you can focus the x-ray beam. With a fine x-ray beam we can look into tissues and cells and see what they contain,” she says.

Says Guerinot, “We can look at one grain of rice and gather a great deal of important information about the plant that produced it.” But the procedure is time-consuming. It takes about 12 hours to scan one virtual slice of an a grain of rice, and several days to produce a full three- dimensional image of numerous scans all stacked up on top of one another. Says Punshon, “We can even do little movies.”

Doing evolution's job

Punshon and Guerinot have demonstrated the power of this technique in another project, using seed from a common weed, called Arabidopsis that is used by researchers because its genome has been mapped. This time, they were interested in learning which genes control how iron, an important nutrient, is taken up by the plant. Punshon explains: “To move mineral nutrients from soil to root, stem or leaf, plants need transporters — shuttles that carry iron to where it is needed for growth.” They were particularly interested in iron transporters that are active in the plant's seed. “Seeds are the most commonly consumed parts of plants. You grind them up for flour, you eat them as corn or peas, you cook them as rice.”

Through genetic experiments they thought they had identified the gene that controls the iron transporter in the Arabidopsis seed. However, when they compared amount of iron in seeds with the gene and seeds without it using a standard chemical analysis, they found no difference. How could that be?

“We suspected that in seeds where the transporter was missing, iron might still be there, but just not where it should be. Which is what our imaging showed." The images showed that in the seeds with the transporter, iron had been shuttled to exactly where it would be needed by the growing plant; in images of seeds without the transporter, the iron was spread around randomly. This technique allowed Punshon and Guerinot to confirm that the gene they had identified was the plant's iron transporter, and that it played a critical role in enabling the plant to load iron onto the seed appropriately.

Knowing where — in what tissues or organs — a gene and its product are expressed provides researchers with important knowledge about a gene's function. Guerinot and Punshon are now starting to image rice grains — using samples from Bangladesh that are high in arsenic — to identify where arsenic accumulates and to help work out which genes they should focus on.

“We already know some of the genes involved,” says Guerinot. The paper published by the Japanese-British team last July describes a pair of transporter genes that move silicon — or its mimic, arsenic — into and out of the plant.

Understanding the whole rice plant at this fine a level of genetic detail will give molecular geneticists and plant breeders the tools they need to solve the arsenic problem. They will have several options. Using genetic engineering, they could knock out certain genes, blocking their function. If these experiments resulted in a genetically modified (or GM) rice whose grains were clean of arsenic, they could offer this new variety quickly to farmers in places such as Bangladesh, where arsenic uptake in rice is a problem and the health risks are immediate and serious. To satisfy farmers whose customers reject GM foods, plant breeders could painstakingly screen existing rice strains for ones in which these same genes are naturally inactive. Through crossbreeding over several growing seasons, they could move those inactive genes into more popular rice varieties.

Likewise, they could create rice varieties — again either through crossbreeding or genetic engineering — in which the function of a gene was enhanced. For instance, they might make a silicon transporter gene more selective, so that it only picked up the less toxic forms of arsenic, or the arsenic forms that are easily excreted by the body, or even so that it couldn't confuse arsenic and silicon at all.

Any of these possibilities could result in a rice plant that can be grown in arsenic-tainted fields, but which would not pass on arsenic to people who eat it. Says Punshon; “We're trying to keep the food supply clean by doing evolution's job for it — only faster.

Recent Publications

Mary Lou Guerinot Pubmed

Tracy Punshon Pubmed

David Salt Pubmed