Secrets of Stress Response

If plants could truly remember, some of us might be in for a coup d'état as our philodendrons finally retaliate after years of neglect. Fortunately, plants don't have consciousness, but they do "remember."

Sally Mackenzie, professor of biology and of plant science, has discovered that the progeny of soybean plants "remember" the stressors--such as drought, extreme cold or heat, and high light levels--of their parents and become more vigorous, resilient, and productive as a result.

Understanding how plants react to environmental stress and how we humans can enhance or direct their responses is a key focus of researchers in the college.

"With regions of the world still food insecure, global population projected to jump from the current 7.7 billion to 9.7 billion by 2050, and climate change expected to make key agricultural areas hotter and dryer, feeding all of humanity will be increasingly challenging," says Gary Thompson, associate dean for research and graduate education.

He notes that the United Nations Food and Agriculture Organization predicts that the global food supply will need to increase by 70 percent to meet rapidly rising demand. "This large expansion in agricultural output will require both improvements in crop yield and the cultivation of additional farmland," he says. "Much of the new agricultural acreage, especially in the developing world, will be arid and semi-arid--marginal for growing crops."

To develop and breed plant varieties that are productive under arduous environmental conditions with little or no added fertilizer, and to enable crops to adapt to climate change and associated extremes of drought, flooding, and temperatures, researchers in the college are probing plant stress responses. By understanding and manipulating the suite of molecular and cellular processes that are triggered by plants' detection of stress, they hope to design crops that can thrive in demanding circumstances.

In the Department of Plant Science, the focus has been on interdisciplinary research that aims to develop stronger and healthier varieties of crops, according to Erin Connolly, professor and department head. The department brings a breadth of expertise to the task in four signature research areas, she says: plant nutrition, soil fertility, and nutrient management; sustainable crop production systems; root/soil processes; and plant stress, climate change, and food security.

Investigating soybean "memory" is one pathway to understanding plant stress related to changing environmental conditions. By temporarily silencing the expression of a critical gene, researchers fooled soybean plants into sensing they were under siege, encountering a wide range of stresses. Then, after selectively cross breeding those plants with the original stock, the progeny "remember" the stress-induced responses to become more vigorous, resilient, and productive plants.

This epigenetic reprogramming of soybean plants, the culmination of a decade-long study, was accomplished not by introducing any new genes but by changing how existing genes are expressed. That is important, says Mackenzie, because it portends how crop yields and tolerance for conditions such as drought and extreme heat will be enhanced in the future.

Mackenzie and her colleagues identified a gene they call MSH1 that exists in all plants, and when they down-regulate or turn off its expression, the plant becomes "convinced" it is encountering multiple stresses, even though it is growing under perfect conditions. The plant senses it is dealing with drought, extreme cold, heat, and high light levels, etc., simultaneously, Mackenzie explains, so it amplifies the expression of gene networks to respond to those stimuli.

Her research group discovered the MSH1 gene more than a decade ago while she was a faculty member at the University of Nebraska-Lincoln studying how genes necessary for energy generation, photosynthesis, and respiration communicate and coordinate. At the time, Mackenzie, now an endowed chair in plant genomics for Penn State's Huck Institutes of the Life Sciences, didn't realize how important the gene is for modifying the way a plant expresses its genes.

"Recently, by serendipity, we discovered that after we replace the MSH1 gene, the plant has a 'memory' of that stress--and by memory I mean its growth features are very different from the plant we started with," she says. "And it will remember the stress generation after generation after generation, as long as we don't make any crosses and keep it in the same lineage."

As part of their research, which was partially supported by grants from the National Science Foundation and The Bill and Melinda Gates Foundation, lines derived from crossing with the "memory" plants were grown in large populations in four different field conditions at four widely separated locations in Nebraska. And they proved to be more vigorous, higher yielding, and better adapted to their environment than typical soybean plants.

Important for the political reality of these times, this is a technology that could be readily applied because it is not a genetically modified organism, so it doesn't require any special regulatory approval. It can go right into the field, Mackenzie points out, and be deployed in any crop, not just in soybean. Her research group has already demonstrated that the approach works in tomatoes and sorghum.

"What it means is that we can take our very best crop varieties and possibly get more out of them and make them more resilient with a fairly straightforward manipulation," she says. "We saw a significant enhancement in yield and growth performance, which is unexpected because we didn't introduce any new genes. We just changed the way they are expressed. And all of a sudden, we had a 13-14 percent increase in the yield of soybeans."

Soybean was a logical crop on which to conduct the research. It is the most widely grown legume in the world, second only to grasses in economic importance. Advances in breeding and agronomic practices have steadily increased soybean yields in the past century, but further improvement will face challenges from climate instability and limited genetic diversity. That calls for the implementation of novel tools and methodologies to benefit soybean performance, researchers say.

The research findings, which were published in Plant Biotechnology Journal, open the door to looking at what epigenetics can offer in the way of crop improvement, Mackenzie believes, "at a time when climate change is going to be the greatest challenge we will deal with over the next 20 to 30 years, and when food security will be very much in jeopardy."

In places like Syria and Lebanon that have been hit so hard by climate change and war that they can't produce their own food, this will be especially important, she notes. "If you start adding up countries that really are not food secure, it is scary. Because if they can't feed their own people, who is going to do it?" she says.

"It is not reasonable to think that we can increase our food production on this continent to manage all of that. One way or another, we have to find ways to produce food in those recalcitrant, difficult environments."

Finding ways to grow crops in harsh environments is a goal of Jonathan Lynch, Distinguished Professor of Plant Nutrition, as well. In the culmination of more than a decade of his research conducted on root traits, about three tons of seed for common bean plants specifically bred to thrive in the barren soils of Mozambique were distributed there last December.

Farmers, nongovernmental organizations, and seed companies in eight villages across the central region of the country in southeast Africa received seed for bean plants that possess an enhanced ability to acquire the essential nutrient phosphorus. The distribution was a joint event led by the Mozambican Institute of Agrarian Research (IIAM), with support from Penn State, the International Center for Tropical Agriculture, the McKnight Foundation, and the U.S. Agency for International Development.

"This is an important milestone, the product of many years of effort here at Penn State in collaboration with our international partners, especially in Mozambique," says Lynch. "With long-term support from our sponsors, we were able to translate scientific discoveries to the practical impact of new bean lines with better stress tolerance."

Because planting season in Mozambique is January and February, the December distribution of seed for more productive bean plants was perfectly timed. Common beans are extremely important as a protein source for smallholder farmers and people living in lower socioeconomic circumstances around the developing world, particularly in southeast Africa, where beans are the primary protein source.

Most soils in Mozambique--one of the poorest nations in Africa, where farmers cannot afford to buy fertilizer if it is even available--are extremely deficient in phosphorus. So developing plants more efficient at taking up the scant available phosphorus is critical. For 11 years or so, Lynch's research group has focused on developing bean plants more adept at taking up phosphorus.

Research has revealed that long and dense root hairs are associated with much more efficient phosphorus uptake, notes Jimmy Burridge, a senior postdoctoral researcher in the Lynch laboratory. And acquiring more phosphorus enables a plant to be more robust and to grow longer roots to follow water down the soil profile.

"For years we have worked to identify traits or characteristics associated with root systems that are related to better performance, and we have passed our findings on to plant breeders, who use them to integrate those root characteristics into new lines," he says. "They have created crosses between varieties with long, dense root hairs and varieties that grow best in Mozambique's harsh conditions."

Studies have shown that bean plants with long, dense root hairs have increased disease tolerance just because they are healthier, Burridge explains. With better nutrition, plants are more energetic--able to grow faster and be more productive.

Penn State's contribution to improving southeast Africa's food-security prospects will endure well beyond the release and distribution of these new bean lines, Lynch points out.

"When we began our work in Mozambique many years ago, there were few trained colleagues in the country, so we first engaged in training several Mozambican scientists, who returned to their posts and are now our partners there," he says.

One, Celestina Jochua, who earned her doctoral degree from Penn State and was advised by Lynch a few years ago, is now working as a plant breeder with IIAM. She spearheaded the effort to develop the new bean lines.

The initiative is one of very few cases in which selection for root traits has been used to improve yield, according to Lynch. There has been considerable scientific interest in deploying root traits in crop breeding because roots are so important for plant growth where drought and low soil fertility are common, but there are few success stories because root biology is complex and challenging.

"The success of this effort shows the way for others to develop more stress-tolerant crops, which are urgently needed in developing countries for food security," Lynch says. "And it's also strategically important for the sustainability and resilience of agriculture in America and other wealthy nations, which confront increasing drought and heat stress from global climate change."

But for now in Mozambique, the new bean lines will have much better yields in stressful conditions than the best lines previously available, so families and communities will have more food security and income, Lynch says.

"This breakthrough shows the value of agricultural research in enabling new technologies that will help us overcome the grand challenge of the twenty-first century--how to sustain almost 10 billion people in a degrading environment."

Not all research so obviously addresses global food insecurity. Some, like that of Surinder Chopra, professor of maize genetics, investigates basic questions in biology with a goal of unraveling mysteries that may eventually lead to resilience against pests and other biotic stressors.

For example, Chopra's most recent discovery of a mutant gene that "turns on" another gene responsible for the red pigments sometimes seen in corn has solved an almost six-decades-old mystery and may have implications for plant breeding in the future.

The culmination of more than 20 years of work, the effort started in 1997 when Copra received seeds from a mutant line of corn. At the time, Chopra was a postdoctoral scholar at Iowa State University, and he brought the research with him when he joined the Penn State faculty in 2000. Much of it has been funded by the National Science Foundation.

The mystery involved a spontaneous gene mutation that causes red pigments to show up in various corn plant tissues, such as kernels, cobs, tassels, silk, and even stalks, for a few generations and then disappear in subsequent progeny. It might seem like a minor concern to the uninitiated, but because corn genetics have long been studied as a model system, the question has significant implications for plant biology.

"In corn, genes involved in pigment biosynthesis have been used in genetic studies for more than a century--pigmentation in corn is a relatively simple trait, which makes it ideal for use as a marker for genetic research," Chopra says. "The mutant corn plants were identified in 1960 by Charles Burnham (University of Minnesota), and that seed was given to one of his students, Derek Styles. We received the seed from Styles in 1997, and we were entrusted to continue the research."

Chopra led efforts to transfer the genes from the mutant corn, dubbed Uf1--Unstable factor for orange1--into various inbred corn lines to be studied. Since he came to Penn State, Chopra's research group has grown and backcrossed lines of corn plants both at the Penn State Agronomy Farm and in greenhouses on campus. In the last three years, the researchers, who published their findings in The Plant Cell, have grown more than 4,000 of the backcrossed plants to map where the cause of Ufo1 is located in the genome.

Using tissues from those hybrid plants, and employing RNA-sequencing techniques and gene-cloning tools along with next-generation sequencing, genetic mapping, and data-analysis capabilities not available to plant geneticists until relatively recently, researchers unmasked the culprit in the on-again, off-again, red-pigment-in-corn mystery. They found Ufo1, which is only present in corn, sorghum, rice, and foxtail millet.

But the Ufo1 mutant gene does not actually cause the red pigments to appear in corn--that is caused by a gene called the pericarp color1, or p1. Researchers found that the Ufo1 gene is actually controlled by a transposon--"jumping gene"--that sits close to the Ufo1 gene. Transposons are sequences of DNA that move from one location in the genome to another, and can influence the expression of nearby essential genes.

When this transposon is switched on, the Ufo1 gene is also turned on, which triggers the p1 gene to signal the plant to produce the red pigments. But when the transposon is off, the Ufo1 gene goes silent and so does the p1-controlled pigment pathway. That is the main reason the Ufo1 gene went unidentified for so long and the mystery persisted, according to Chopra.

"We were able to narrow it down to a single gene out of several thousand genes that are aberrantly expressed in the Ufo1 mutant versus the wild-type plant," he says. "It is an incremental discovery, and yet it is a leap in basic science because it is likely to be valuable to plant breeders."

It is still not entirely clear how Ufo1 interacts with the p1 gene. The discovery's future significance likely will be less associated with red pigments than what the Ufo1 mutant gene controls in corn plants. Chopra believes it may be a "master regulator" that, when overexpressed, signals the plant that it is under stress, even in the absence of stress.

Interestingly, Chopra points out, in Ufo1 plants, sugars overaccumulate in leaves, and the content of maysin, a natural insecticide made by corn plants, sharply increases in the silk.

"Learning about what controls the regulation of the normal or the nonmutant Ufo1 gene will bring us much closer to a realistic breeding process in which we can tinker with gene expression to get higher maysin content or increased sugar content, which would be important in crop protection from pests and biofuel production, respectively," Chopra says.

"And, because it has a pronounced effect on the workings of the cellular machinery, we can now understand further the basic molecular pathway that normally happens during a stress to a plant," he says. "Understanding plant stress resulting from extremes of heat, cold, and water is important because of climate change."

To broaden its plant stress research effort, the Department of Plant Science is developing a formal partnership with the University of Nottingham in the United Kingdom to conduct research related to its "Future Food/Beacon of Excellence" program. Nottingham's venture aims to address world hunger by developing new, resilient crops that are nutrient rich.

More specifically, Penn State and Nottingham will capitalize on research and infrastructure strengths to study the connection between crop quality and health outcomes, Connolly noted. "In Africa, for example, millions of people suffer from iron deficiency, which can lead to poor health outcomes," she says. "Developing crops that are rich in iron--and can grow with minimal resources--can help to improve the health of communities."

But the effort to understand and harness plants' reaction to stress in the college is bigger than one department, Thompson says. Related research is also being conducted in the Departments of Plant Pathology and Environmental Microbiology and Ecosystem Science and Management, dealing with biotic stressors from disease to pests.

"It is a growing strength in the college to consider stressors from a number of angles and at different scales--from the microbiome to the ecology of ecosystems," he says. "There can be no single solution to a challenge as big as food security, and we will need as many possible methods of addressing the relative unpredictability of climate change impact as we can develop."

By Jeff Mulhollem
Illustrations by Jonathan Carlson