Purdue News

September 1, 2004

Purdue study finds antioxidant protects metal-eating plants

WEST LAFAYETTE, Ind. - An antioxidant, a type of compound that prevents certain types of damage to living cells, appears to allow some kinds of plants to thrive on metal-enriched soils that typically kill other plants, says a Purdue University scientist.

David Salt (R), John Freeman
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This finding, published in the current issue of The Plant Cell, provides an important new insight for the development of plants that could be used to help clean polluted sites. The work also answers a fundamental question for researchers studying how certain types of plants tolerate levels of metals in their tissues that are toxic to most other plants.

"We were able to clearly establish for the first time that plants that create and accumulate high cellular levels of the antioxidant glutathione are much more nickel tolerant," said David Salt, associate professor of plant molecular physiology in Purdue's horticulture department.

The term antioxidant generally refers to a broad class of compounds that protect cells from damage otherwise caused by exposure to certain highly reactive compounds.

Understanding the mechanism behind nickel tolerance provides an important tool for researchers like Salt, whose goal is to develop plants that remove toxic metals from the environment in a process known as phytoremediation, or extract useful metals from soil, a process known as phytomining.

While previous research has shown where metals reside in a plant's cell, this is some of the first data showing how plants protect themselves from the damaging effects of those metals.

"One major hurdle to developing hyperaccumulating plants is toxicity," said John Freeman, first author of the paper and a doctoral student working with Salt. "For a plant to hyperaccumulate metal, it has to be able to tolerate metal toxicity."

A nearly ubiquitous antioxidant, glutathione plays a critical role in minimizing oxidative stress, or damage caused by highly reactive compounds, Salt said.

Plants require metals like nickel in minute quantities for certain metabolic processes, but at high levels metals can damage membranes, DNA and other cell components. Most plants try to keep the levels of metals in their cells at a minimum, but plants called metal hyperaccumulators have the unique ability to build up unusually high levels of metals in their tissues without any ill effect.

Previous research indicates that hyperaccumulators store metals in a specialized cell compartment called the vacuole. Sequestered in the vacuole, nickel and other metals can't damage other parts of the cell. But nickel still must travel within the cell in order to enter the vacuole in the first place, Salt said.

"To get to the vacuole, the nickel has to traverse the interior of the cell, where most of the plant's sensitive biochemical processes reside," he said. "So we've been interested in finding out if there's something in the cell's interior that protects it from oxidative damage as the metal crosses the cell."

In this study, Salt and his colleagues sampled a number of closely related plants that grow on soils naturally enriched in nickel. These plants ranged from those that didn't accumulate any nickel to the hperaccumulators that built up almost 3 percent nickel by weight.

He found that the concentration of glutathione was well correlated with a plant's ability to accumulate nickel.

"This correlation makes good sense," Salt said. "If you accumulate a lot of nickel, then you will need the ability to resist high levels of oxidative stress."

Correlation doesn't prove causation, however, so the next step in Salt's study was to establish that glutathione played a functional role in nickel tolerance.

He and his colleagues isolated a gene called SAT, and inserted it into a model lab plant called Arabidopsis thaliana, which does not normally tolerate nickel. The gene SAT produces an enzyme called serine acetyltransferase, which plays a role in producing glutathione in hyperaccumulating plants.

When Salt grew both normal Arabidopsis and those containing the SAT gene on a nickel-containing medium, the normal plants failed to grow and showed signs of severe membrane damage, an indicator of oxidative stress. The plants with the inserted gene thrived, showing no signs of membrane damage.

Going one step further, Salt conducted another experiment in which he exposed the Arabidopsis containing the SAT gene to a compound that inhibits their ability to make glutathione. When grown on nickel, these plants also suffered high levels of oxidative damage, just like their normal counterparts.

"This confirms that it really is glutathione that's responsible for nickel tolerance," Freeman said.

This research is part of a larger gene discovery initiative involving Purdue's Center for Phytoremediation Research and Development, a multidisciplinary research center dedicated to developing a "molecular toolbox" that will provide the genetic information to develop plants ideally suited to the phytoremediation of polluted sites. Technologies developed at the center will be commercialized through a partnership with the Midwest Hazardous Substance Research Center, a U.S. Environmental Protection Agency regional hazardous substance research center.

Also participating in this project were undergraduate student Ken Nieman, technician Carrie Albrecht and research scientist Wendy Peer of Purdue; Michael W. Persans, who was a postdoctoral scientist in Salt's laboratory and now has a position at the University of Texas Pan-American; and Ingrid J. Pickering of the Stanford Synchrotron Radiation Laboratoty. Funding was provided by The National Science Foundation, the U.S. Department of Energy and the National Institutes of Health.

Writer: Jennifer Cutraro, (765) 496-2050, jcutraro@purdue.edu

Sources: David Salt, (765) 496-6454, salt@hort.purdue.edu

John Freeman, (765) 494-9398, jlfreema@purdue.edu

Ag Communications: (765) 494-2722; Beth Forbes, forbes@purdue.edu
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David Salt, at right, a Purdue University professor of horticulture, and doctoral student John Freeman are finding out why certain plants thrive while others wither when exposed to massive amounts of nickel. Their findings provide insight for the development of plants that can remove toxic metals from polluted sites or extract useful metals from soil, a process known as phytomining. (Purdue Agricultural Communications photo/Tom Campbell)

A publication-quality photograph is available at http://ftp.purdue.edu/pub/uns/+2004/salt-antiox.jpg



Increased glutathione biosynthesis plays a role in nickel tolerance in Thlaspi nickel hyperaccumulators

John L. Freeman, Michael Persans, Ken Nieman, Carrie Albrecht, Wendy Peer, Ingrid J. Pickering, and David E. Salt

Worldwide, more than 400 plant species are now known that hyperaccumulate various trace metals (Cd, Co, Cu, Mn, Ni, and Zn), metalloids (As), and nonmetals (Se) in their shoots. Of these, almost one-quarter are Brassicaceae family members, including numerous Thlaspi species that hyperaccumulate Ni up to 3% of their shoot dry weight. We observed that concentrations of glutathione, Cys, and O-acetyl L-serine (OAS), in shoot tissue, are strongly correlated with the ability to hyperaccumulate Ni in various Thlaspi hyperaccumulators collected from serpentine soils, including Thlaspi goesingense, T. oxyceras, and T. rosulare, and nonaccumulator relatives, including T. perfoliatum, T. arvense, and Arabidopsis thaliana. Further analysis of the Austrian Ni hyperaccumulator T. goesingense revealed that the high concentrations of OAS, Cys, and GSH observed in this hyperaccumulator coincide with constitutively high activity of both seine acetyltransferase (SAT) and glutathione reductase. SAT catalyzes the acetylation of L-Ser to produce OAS, which acts as both a key positive regulator of sulfur assimilation and forms the carbon skeleton for Cys biosynthesis. These changes in Cys and GSH metabolism also coincide with the ability of T. goesingense to both hyperaccumulate Ni and resist its damaging oxidative effects. Overproduction of T. goesingense SAT in the nonaccumulator Brassicacear family member Arabidopsis was found to cause accumulation of OAS, Cys, and glutathione, mimicking the biochemical changes observed in the Ni hyperaccumulators. In these transgenic Arabidopsis, glutathione concentrations strongly correlate with increased resistance to both the growth inhibitory and oxidative stress induced effects of Ni. Taken together, such evidence supports out conclusion that elevated GSH concentrations, driven by constitutively elevated SAT activity, are involved in conferring tolerance to Ni-induced oxidative stress in Thlaspi hyperaccumulators.


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