A group of plants that uses metal to defend  against infection may do so because the normal defense mechanism used by most other plants is blocked.

Purdue University researchers found that this group of plants produces, but does not respond to, the molecule that triggers the infection response used by nearly all other plants. The molecule does, however, allow this group of plants, called metal hyperaccumulators, to store high levels of metal in their tissues, rendering them pathogen
resistant.

These findings, reported in today's issue of the journal Plant Physiology, shed new light on the evolution of these plants and may have implications for the development of crops that may one day remove metal and other contaminants from the environment.

"Our goal is to find the high-level regulator - the one gene or group of genes that turns a plant into a hyperaccumulator," said David Salt, associate professor of plant molecular physiology in Purdue's horticulture department. "But we have no way to know what that gene is, so we need to deconstruct the process, starting with things we can measure, which are these visible traits, and then we work backwards."

A question that has long stymied Salt and his colleagues centers on the origins of this trait. While essential as micronutrients, metals are toxic in high levels. Most plants have mechanisms that keep metals in the environment out of their tissues. So what would have driven some plants to do just the opposite?

"The existing explanation is that metal accumulation evolved to protect these plants from pathogens," Salt said. "Yet most other plants don't accumulate metals, and they resist infection just fine. It never really made sense to me. If everyone's already resisting pathogens, why do you need an extra mechanism? There has to be more to it."

It turns out the plants Salt studies - a group of small, weedy alpine flowers called Thlaspi - lack the standard pathogen defense mechanism found in nearly every other plant species. Thlaspi plants live in soils naturally enriched in nickel, and when growing in their natural habitat, are not any more susceptible to pathogens than similar plants growing nearby.

When grown in the absence of metal, however, these plants are defenseless against diseases like powdery mildew, a common fungal infection that most other plants fight off with ease.

In most plants, exposure to powdery mildew and other pathogens triggers the plant defense pathway, a series of biochemical events that occur in succession and help the plant resist infection. A molecule called salicylic acid - a common plant compound and the active ingredient in pharmaceuticals like aspirin and acne medications - governs this pathway.

When faced with a fungus or bacteria, most plants turn up their production of salicylic acid, which then interacts with other molecules in the plant, eventually turning on the genes that produce the proteins involved in fighting infection. These infection-fighting proteins also turn off salicylic acid production, a phenomenon known as negative feedback. In this way, plants can turn the pathogen defense pathway on and off as needed.

Most plants maintain very low levels of salicylic acid in their tissues unless they are fighting an infection. Metal hyperaccumulators, however, have significantly elevated salicylic acid in their tissues all the time.

In the current study, Salt and his colleagues compared salicylic acid levels in both the hyperaccumulator Thlaspi and the common lab plant Arabadopsis, which does not accumulate metal. They also compared fungal infection rates in both types of plants when grown with or without exposure to the metal nickel.

They found significantly higher levels of salicylic acid in the hyperaccumulator compared to the non-accumulator. In addition, while Thlaspi thrived in metal-enriched soil, it succumbed to a severe fungal infection when no metal was present.

"This difference in salicylic acid levels raises several questions," Salt said. "If you modify other plants so that the level of salicylic acid is always high, those plants are not happy. They look sickly. With salicylic acid continuously elevated, a plant thinks it's under some massive attack by a pathogen. It's expressing all its pathogen response
proteins, and at such a high level, they can have a deleterious effect on the plant."

Metal hyperaccumulators like Thlaspi, however, don't show any negative effects from their constant exposure to high levels of salicylic acid.

"These plants have tons of salicylic acid, but for some reason that salicylic acid is not initiating the pathogen response. That tells us some part of the pathway doesn't sense salicylic acid - that the signal is blocked," he said. "It's like yelling into the phone louder and louder, but no one can hear it."

Salt and his colleagues also show in the current study that salicylic acid induces production of a molecule called glutathione, a potent antioxidant that protects plants from metal. Because the production of glutathione is tied to the production of salicylic acid, most plants normally have fairly low glutathione levels and, consequently, can't
tolerate metals.

Thlaspi, on the other hand, is brimming with glutathione, thanks to its elevated salicylic acid levels. When grown in nickel-enriched soil, Thlaspi takes up 3 percent of its body weight in the metal. Salt and his colleagues have shown that this metal content is what makes the plants resistant to pathogens.

Salt proposes a scenario in which at some point in evolutionary history some plants acquired a mutation that disrupts the salicylic acid signaling pathway, leaving them unable to fight off pathogens.

"In most settings, those plants would be toast - they'd be immediately selected out of the population," he said. "This whole system raises the question of evolution.

"But let's say, by some obscure chance, those plants were growing on soils with elevated metals. We've shown that high salicylic acid levels produce high glutathione levels. We know high glutathione is crucial for nickel tolerance, and when this plant accumulates nickel, it becomes pathogen resistant. So now the plant doesn't die; it can propagate, and over time this can evolve as a more enhanced system."

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" to provide the genetic information to develop plants to clean up 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.

Salt collaborated in this research with John Freeman, a former graduate student now at the University of Colorado, Fort Collins. Graduate students Daniel Garcia and Amber Hopf and postdoctoral scientist Donggium Kim at Purdue's Center for Plant Environmental Stress Physiology also participated in this research. The National Science Foundation and the Indiana 21st Century Research and Technology Fund funded this project, with support from the Bindley Bioscience Center in Purdue's Discovery Park.

Related Web sites:

Purdue Center for Plant Environmental Stress Physiology: http://www.hort.purdue.edu/cfpesp/cfpesp.htm

Purdue Bindley Bioscience Center:
http://discoverypark.e-enterprise.purdue.edu/wps/portal/.cmd/cs/.ce/155/.s/4283/_s.155/4283

ABSTRACT

Constitutively Elevated Salicylic Acid Signals Glutathione-Mediated Nickel Tolerance in Thlaspi Nickel Hyperaccumulators
John L. Freeman, Daniel Garcia, Donggiun Kim, Amber Hopf, and David E. Salt

Progress is being made in understanding the biochemical and molecular basis of nickel (Ni)/zinc (Zn) hyperaccumulation in Thlaspi; however, the molecular signaling pathways that control these mechanisms are not
understood. We observed that elevated concentrations of salicylic acid (SA), a molecule known to be involved in signaling induced pathogen defense responses in plants, is a strong predictor of Ni hyperaccumulation in the six diverse Thlaspi species investigated, including the hyperaccumulators Thlaspi goesingense, Thlaspi rosulare,
Thlaspi oxyceras, and Thlaspi caerulescens and the nonaccumulators Thlaspi arvense and Thlaspi perfoliatum. Furthermore, the SA metabolites phenylalanine, cinnamic acid, salicyloyl-glucose, and catechol are also elevated in the hyperaccumulator T. goesingense when compared to the nonaccumulators Arabidopsis (Arabidopsis thaliana) and T. arvense. Elevation of free SA levels in Arabidopsis, both genetically and by exogenous feeding, enhances the specific activity of serine acetyltransferase, leading to elevated glutathione and increased Ni resistance. Such Samediated Ni resistance in Arabidopsis phenocopies the glutathione-based Ni tolerance previously observed in Thlaspi, suggesting a biochemical linkage between SA and Ni tolerance in this genus. Intriguingly, the hyperaccumulator T. goesingense also shows enhanced sensitivity to the pathogen powdery mildew (Erysiphe cruciferarum) and fails to induce SA biosynthesis after infection. Nickel hyperaccumulation reverses this pathogen hypersensitivity, suggesting that the interaction between pathogen resistance and Ni tolerance and hyperaccumulation may have played a critical role in the evolution of metal hyperaccumulation in the Thlaspi genus.