In Fight Against Pathogens, Calcium Helps Plants Make Their Own Aspirin

Calcium builds strong bones, good teeth - and healthy plants, according to a new study from Washington State University.

Experiments show that calcium, when bound to a protein called calmodulin, prompts plants to make salicylic acid (SA) when threatened by infection or other danger. SA is a close chemical relative of aspirin. In plants, SA acts as a signal molecule that kicks off a series of reactions that help defend against external threats.

"That plants make salicylic acid has been known for more than 100 years," said B.W. Poovaiah, Regents Professor and director of the study, "but the role of calcium in signaling a plant to make SA has not been known before."

"We are now beginning to understand the molecular mechanism connecting the calcium/calmodulin signaling to plant immunity," said Poovaiah.

The study will appear in an upcoming issue of the journal Nature and is now available online at http://dx.doi.org/10.1038/nature07612.

Poovaiah said that in controlling salicylic acid level, calcium acts like a gatekeeper within the cells of a plant, directing incoming information and helping the plant respond to such dangers as pathogen attacks. Normal, healthy plants have a low level of SA in their cells. That level rises when the plant is threatened by infection or environmental stress.

"When we expect danger, we try to take precautions," said Poovaiah. "Plants cannot run away. Plants have to turn on their built-in system to protect themselves. The plant has to produce different signal molecules. One of them is salicylic acid."

According to lead author, assistant research professor Liqun Du, SA sets off defensive measures within the plant, such as the "hypersensitive reaction" in which cells around an infection site die, forming a barrier that keeps the pathogen from invading deeper into the plant. That provides protection against the immediate threat. SA also activates the plant's systemic acquired resistance, a form of immunity that protects the plant from further pathogen attacks.

But a rise in SA levels also causes the plant to slow its growth, perhaps saving its strength for the battle against the pathogen. That sets up a challenging situation for both the plant - grow faster or protect myself better? - and farmers, who might view SA as a tool to protect their plants from disease. A plant that makes high levels of SA all the time will be safe from infection but will grow slowly. A plant that makes little or no SA will grow like gangbusters but be very susceptible to infection.

"It's a fine balance," said Du. "Too much is bad; too little is bad."

Working with the small mustard plant Arabidopsis thaliana, Poovaiah¹s research team showed that the key step in this balancing act is the interaction between calcium/calmodulin and a protein called AtSR1, which suppresses the production of salicylic acid. Stress or infection causes a spike in calcium within the plant cells, which, in combination with calmodulin, acts as a specific signal controlling the activity of AtSR1 and the formation of SA.

In an environment with few pathogens, a plant will have low levels of SA. The plant lets its guard down and devotes more resources to growth. If it becomes infected, SA production goes up, and the plant dials back its growth and puts more resources into defense.

The importance of AtSR1 was especially clear in experiments in which plants were engineered to have more or less AtSR1 than normal. Plants that have extra AtSR1 make almost no SA. They grow larger and faster than a normal plant, but easily succumb to infection. Plants that lack the gene for AtSR1 develop high levels of SA and deploy their immune responses all the time. They are nearly impervious to infection, but small in size. The same is true of plants whose AtSR1 has been changed so it cannot bind to calcium/calmodulin, demonstrating the crucial role played in this system by calcium and calmodulin.

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Scientists Discover an Ancient Odor-Detecting Mechanism in Insects

Newly discovered receptors in the nose of a common insect unearths one of the most ancient mechanisms that organisms have evolved to smell. Ionotropic glutamate receptors, proteins that reside deep in the brain at the synapses, grab glutamate molecules and quickly relay messages from one nerve cell to the next, helping animals learn, move and remember. But insects do not relegate these receptors to the depths of the brain. They also put them to use elsewhere: in the nose.

"On the surface it's a completely absurd idea," says Leslie B. Vosshall, head of the Laboratory of Neurogenetics and Behavior at Rockafeller University. "We know what these proteins do; they sit at the synapse and mediate fast neuronal communication. So the idea that the fly has massively expanded the number of these receptors and positioned them to interact with small molecules in the air seems very strange. But if you think about it, it makes sense. The process is the same, but rather than grabbing small molecules at the synapse, they're grabbing small molecules from the air."

The project began two years ago, when Vosshall and Richard Benton, then a postdoc in her lab, noticed a group of six ionotropic glutamate receptor genes while sifting through the fly genome. Although this group was recognized 10 years ago, ever since the genome was sequenced, the genes did not have a known function, in part because it was assumed they must be similar to any other ionotropic glutamate receptor deep in the fly brain. But to Vosshall and Benton, who is now at the Center for Integrative Genomics in Lausanne, Switzerland, that didn't matter.

Vosshall and her team wondered whether these receptors could in fact represent the "missing" receptors thought to exist in the fly's "nose" -- its two antennae. Each antenna is divided into three types of smell neurons. Scientists have characterized the receptors that detect odors in two of these types but those receptors were mysteriously absent in the third, a swath of territory known as the coeloconic sensilla. "It has been shown that cells in the coeloconic sensilla detect odors," Vosshall says. "It's just that we didn't know how they did it."

The team showed that these receptors, which the Vosshall lab named ionotropic receptors, do in fact explain how cells in coeloconic sensilla detect odors. First, they showed that they are expressed in complex combinatorial patterns at the sensory end of olfactory neurons where they have access to and can scan the outside world for odors. They then showed that when these receptors are expressed in the cells in the coeloconic sensilla, the cells respond to odors. Finally, the researchers showed that when they plucked a receptor -- say one that detects an odor that resembles a mix of grass and honey -- out of its native cell and genetically embedded it in a different cell, the new cell would now detect that odor.

Although it is still unclear why insects have developed two sets of chemosensory receptors -- olfactory receptors and ionotropic receptors -- the work raises questions regarding their evolutionary origin. Ten years ago, researchers at New York University revealed that plants, which detect soil nutrients and chemicals in the air, also express glutamate receptors, suggesting that the ancestral origin of glutamate receptors may have been to detect small molecules in the air, rather than small molecules in the brain.

"In a way, these receptors were very well hidden because everyone assumed that they were extra glutamate receptors that were unlikely to be of interest," explains Vosshall. "All we did to find them was searched for a gene family of unknown function -- and left our preconceived notions aside."

This work was funded in part by grants from the Foundation for the National Institutes of Health through the Grand Challenges in Global Health Initiative and the National Institutes of Health.

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Scientists Pull Genetic Tail to Curtail Cancer

>An innovative researcher followed a hunch and encouraged activity in an important cellular tumor-suppressor by grabbing it by the tail.

Inside human cancer cells, the important tumor-suppressing protein often seems to play hooky, lolling around in cellular broth instead of muscling its way out to the cells' membranes, where it can prevent cancer growth. This delinquency puzzled scientists for a long time, until a cell biologist in the Johns Hopkins University School of Medicine felt compelled to genetically grab the protein by the tail and then watched as it got back to work at tamping down disease.

"It was curious that when we removed its tail, the protein suddenly was unhindered and moved out to the membrane and became active," says Meghdad Rahdar, a graduate student in pharmacology.

The discovery, published Dec. 15 online at the Proceedings of the National Academy of Sciences, represents a potential new approach to cancer therapy, according to Peter Devreotes, Ph.D., professor and director of cell biology at Johns Hopkins.

"A long-term goal is to find a drug that does the equivalent of our bit of genetic engineering," he says.

The flexible tail contains a cluster of four amino acids ‹ the building blocks of proteins ‹ that regulate this tumor suppressor known as PTEN. When chemically modified, these amino acids act to ³glue² the tail back to the body of PTEN and prevent the attachment of PTEN to the membrane. By genetically removing PTEN's tail, or manipulating the cluster of four amino acids so that they cannot be modified, the researchers persuaded PTEN to move to the cell membrane where it goes about its tumor-suppressing business of degrading a molecular signal called PIP3 that causes errant cell growth.

"As far as I know, I haven't seen anyone activate a tumor suppressor, but we seem to have done it genetically," Rahdar says.

While genetically engineering cancer cells in the human body is neither practical nor safe, manipulating such unbinding of PTEN with drugs is a viable alternative to guard against cell overgrowth, the hallmark of cancer, the Hopkins scientists say.

In many tumors, PTEN is simply not present. In others, it's there, but a key enzyme that produces PIP3 is over-activated. The Hopkins team already has shown the first evidence that adding the modified PTEN to cells that lack PTEN not only restores normal enzyme levels but ramps up PTEN activity and quells the cell growth signal.

The research was supported by the National Institutes of Health.

In addition to Rahdar and Devreotes, authors on the paper are Takanari Inoue, Tobias Meyer, Jin Zhang and Francisca Vazquez, all of Johns Hopkins.

On the Web:

http://www.hopkinsmedicine.org/cellbio/devreotes http://www.pnas.org

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Grape Seed Extract Kills Laboratory Leukemia Cells, Proving Value of Natural Compounds

An extract from grape seeds forces laboratory leukemia cells to commit cell suicide. Within 24 hours of exposure to the naturally occurring compound, 76 percent of leukemia cells had died. The grape seed extract that led to cell death, or apoptosis, by activating JNK, a protein that regulates the apoptotic pathway.

"These results could have implications for the incorporation of agents such as grape seed extract into prevention or treatment of hematological malignancies and possibly other cancers," said the study's lead author, Xianglin Shi, Ph.D., professor in the Graduate Center for Toxicology at the University of Kentucky.

"What everyone seeks is an agent that has an effect on cancer cells but leaves normal cells alone, and this shows that grape seed extract fits into this category," he said.

Shi adds, however, that the research is not far enough along to suggest that people should eat grapes, grape seeds, or grape skin in excess to stave off cancer. "This is very promising research, but it is too early to say this is chemo-protective."

Hematological cancers ­ leukemia, lymphoma and myeloma ­ accounted for an estimated 118,310 new cancer cases and almost 54,000 deaths in 2006, ranking these cancers as the fourth leading cause of cancer incidence and death in the U.S.

Given that epidemiological evidence shows that eating vegetables and fruits helps prevent cancer development, Shi and his colleagues have been studying chemicals known as proanthocyanidins in fruits that contribute to this effect. Shi has found that apple peel extract contains these flavonoids, which have antioxidant activity, and which cause apoptosis in several cancer cell lines but not in normal cells. Based on those studies, and findings from other researchers that grape seed extract reduces breast tumors in rats and skin tumors in mice, they looked at the effect of the compound in leukemia cells.

Using a commercially available grape seed extract, Shi exposed leukemia cells to the extract in different doses and found the marked effect in causing apoptosis in these cells at one of the higher doses.

They also discovered that the extract does not affect normal cells, although they don¹t know why.

The researchers then used pharmacologic and genetic approaches to determine how the extract induced apoptosis. They found that the extract strongly activated the JNK pathway, which then led to up-regulation of Cip/p21, which controls the cell cycle.

They checked this finding by using an agent that inhibited JNK, and found that the extract was ineffective. Using a genetic approach ­ silencing the JNK gene ­ also disarmed grape seed extract's lethal attack in leukemia cells.

"This is a natural compound that appears to have relatively important properties" Shi said.

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Secrets of Pheromone Detection

Newswise — Researchers at UT Southwestern Medical Center have overturned the current theory of how a pheromone works at the molecular level to trigger behavior in fruit flies.

The finding, if it proves true in other species, might lead to new ways to manipulate the actions of harmful insects, the researchers said.

They found that the pheromone, which affects mate recognition and sexual behavior, does not directly attach to nerve cells, as was previously thought. Rather, the pheromone first docks onto a free-floating protein in the insect’s antennae called LUSH.

This docking action causes LUSH to change shape. The reshaped protein, not the pheromone itself, is what activates the nerve cells, the researchers found. Their findings show that the pheromone only indirectly controls the animal’s behavior.

“If we can inhibit this molecular interaction, we might be better able to control pests that harm crops, carry malaria and so forth,” said Dr. Dean Smith, associate professor of pharmacology and neuroscience at UT Southwestern and senior author of the study, which appears online today and in Friday’s issue of the journal Cell.

Pheromones are molecules that an organism releases to trigger a specific behavior in other members of its species. Insects make wide use of pheromones to attract mates, signal the location of food, warn of attackers and provide other signals. Detection of pheromones is so sensitive that, in some cases, a single molecule is enough to trigger a response.

In agriculture, pheromones for some pest species are already used to protect crops by disrupting reproduction.

The current study used the fruit fly Drosophila melanogaster and focused on a pheromone called cVA (11-cis vaccenyl acetate). Only male flies produce cVA, but both males and females react to it. It is involved in clustering, mate recognition and sexual behavior.

The researchers examined the interaction of cVA with a type of nerve cell called T1 in the antennae of Drosophila. Holes in the antenna allow cVA to enter a fluid-filled chamber that surrounds these nerve cells. When the pheromone enters the antenna, the nerve cells fire.

The fluid surrounding the nerve cells also contains the LUSH protein, which was already known to bind to cVA. The prevailing theory is that LUSH proteins act as a carrier, picking up any cVA molecules that enter the antenna, transporting them to the nerve cells and releasing the pheromone molecules to bind directly to the nerve cells, Dr. Smith said.

In their experiments, Dr. Smith’s group examined the nerve cells’ firing patterns in genetically altered flies. The researchers also created variations of LUSH with varying abilities to change shape. One variant of LUSH, designed to mimic the shape it takes when bound to cVA, was capable of stimulating the nerve in the complete absence of the pheromone.

“LUSH is clearly important for activating the nerve,” Dr. Smith said. “The nerve cell’s receptor is designed to work only when cVA and LUSH are bound together. It’s a very unusual pathway that allows even single pheromone molecules to activate the nerves.”

The researchers are further examining the interaction between the pheromone and the nerve cell receptors.

Dr. Smith said if pheromone systems work this way in other insect species, researchers might be able to design compounds that block the action of pheromones, allowing them to manipulate and control pest behavior, including controlling mating and reproduction.

Dr. Tal Soo Ha, postdoctoral researcher in pharmacology at UT Southwestern, and researchers from the University of Colorado Denver also participated in the study.

The work was funded by the National Institutes of Health, with further support from the Howard Hughes Medical Institute and the University of Colorado Cancer Center.

Dr. Dean Smith -- http://www.utsouthwestern.edu/findfac/professional/0,2356,16780,00.html

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