Inside Salk; Salk Insitute

Seeds of change

California’s bristlecone pines are some of the oldest trees in the world.

High in the White Mountains of California, anchored to a rocky slope by its gnarled roots, is a tree older than Methuselah.

Now knotted and twisted with age, the tree sprouted from seed hundreds of years before the Egyptians built the great pyramids. It was roughly 3,000 years old when Julius Caesar was born and 4,000 years old when Genghis Kahn ruled the Mongol Empire. The ancient arbor, a bristlecone pine, predates its famous California neighbor, “Methuselah,” another bristlecone named after the Bible’s longest-lived man and once thought to be the oldest tree in the world.

Over its 5,064-year lifespan, the pine has experienced droughts as bad, if not worse, than the one currently parching California and other parts of the American West. It has lived through long snowy winters, insect invasions, lightning storms, raging forest fires, torrential rains–a litany of ordeals. The tree’s life is an epic tale, but what can we humans learn from a weathered old tree? Perhaps the most poignant lesson is this: to stay in one place requires remarkable flexibility. To survive is to adapt.

“With our nomadic tendencies, we rely on our mobility for our survival,” says Joanne Chory, director of Salk’s Plant Molecular and Cellular Biology lab. “If our current location gets dicey, we can make tracks for another. For plants, though, location is destiny. Because of this, they have developed a wide range of tools for adjusting to whatever their environment throws at them.”

Jianyan Huang observes seedlings grown in a high-light chamber. Her research could help develop new high-light tolerant crop varieties.

It is these tools, a range of genetic programs, molecular devices and versatile chemistry plants deploy like the blades of a Swiss Army knife, that Chory and other Salk plant biologists are intent on cataloging and describing. In this quest, they are propelled by curiosity, the scientist’s driving force. But another powerful motivation is necessity–the need to ensure the longevity of Homo sapiens.

Crunch Time for Farmers

To understand what’s at stake, it helps to consider that the global human population recently topped 7 billion and is expected to reach 10 billion in just 35 years. This population crush equates to crunch time for farmers. More people means surging demand for the myriad of products plants provide, from food on the table to the shirts on our backs. United Nations experts predict that agricultural yield must double in the next two decades to meet the demands for natural resources. And as the pressure on farms constantly increases, weather patterns are increasingly inconstant. Thanks to global climate changes, some regions are experiencing extreme drought and desertification, while others are experiencing violent storms and flooding.

“In California right now, everyone’s focused on the drought, and for good reason, but water stress is only one kind of stress plants experience,” says Chory, who is also a Howard Hughes Medical Institute Investigator and holder of Salk’s Howard H. and Maryam R. Newman Chair in Plant Biology. “These old bristlecone pines, exposed on the side of a mountain, have seen extremes of all sorts–really hot, really cold, lots of water, no water.

“Agricultural crops grow in more moderate conditions,” she adds, “but they still have to cope with various kinds of stressors, and farmers have to make sure they plant the right varieties for the climate, or they’ll end up with a poor yield or dead crops. With rapid climate change, a great challenge is for our agricultural practices to keep up with environmental extremes.”

Joanne Chory

This is where science can help. One of the great breakthroughs in science was the discovery that when farmers breed for certain desirable characteristics, they are selecting for certain genetic patterns. A recent study published in the journal Cell, for instance, identified a specific gene, COLD1, that confers cold tolerance to domesticated rice grown in Japan, where temperatures can be too chilly for wild rice species. Finding a gene that helps a plant survive cold stress helps explain what was going on under the hood as farmers drove rice cultivation further north. It might also help in the development of new varieties of cold-tolerant rice or convey cold tolerance to other crops through genetic engineering.

As Chory points out, a single plant can experience a range of extremes over its lifetime, or even over the course of a year. Joseph Noel, another Salk plant biologist and longtime collaborator with Chory, notes that there are trade-offs in a plant’s ability to tolerate certain types of stress.

Joseph Noel

“A plant that is highly drought resistant isn’t going to grow as well in a rainy year,” says Noel, holder of Salk’s Arthur and Julie Woodrow Chair and also a Howard Hughes Medical Institute Investigator. “Ideally, a farmer would be able to look at the climate forecast for the upcoming growing season–which have gotten much more accurate–and pick a crop variety that’s optimized for the predicted weather. It would work sort of like getting a yearly flu shot, where the vaccines are developed based on informed predictions. It won’t work all the time, but on average, you’d come out with better results and more sustainable crop yields.”

Plants, Proteins and Possibilities

In a new line of research, Noel’s lab is exploring a related concept: how plants respond to subtle variations in drought. Specifically, he’s studying a plant hormone called abscisic acid (ABA) that, among other things, turns on a plant’s emergency response program during drought. When the roots sense the soil is drying out, they release ABA, which travels through the stems to the leaves. There, the hormone closes the stomata, the openings in the leaves that exchange oxygen for carbon dioxide. This conserves the plant’s water stores, as water vapor can escape from the open stomata. Scientists knew that ABA signaled the stomata to close by flipping molecular switches known as PYR/PYL receptors,14 varieties of which are found in the Arabidopsis thaliana, a mustard plant that serves as a model for plants in laboratory research. Initially, scientists thought the interactions seemed to be all-or-nothing in that ABA bound all versions of the receptor, switching them all on when it was present. In recent studies however, Noel’s team believes they’ve uncovered a more complex relationship, one in which alternate forms of the ABA bind some, but not all, of the PYR/PYL receptors, and bind some receptors more tightly than others.

“Looking at this through the lens of evolution and adaptation, it could mean that this gives you a range of different responses to dry soil,” says Noel. “We thought the ABA drought response function was an on-off switch, but maybe it’s more like a dimmer switch, where you can dial in a precise response to water stress. Back to that idea of precision agriculture: the farmer could pick the appropriate variety of plant based on its ABA profile and match it to climate forecasts.”

In another project, Charisse Crenshaw, a postdoctoral researcher in Noel’s lab, has teamed up with Salk Associate Professor Tatyana Sharpee, an expert in computational biology and neuroscience, to study the flexibility of a plant’s genome in responding to its environment. Plants have apparently repurposed a few basic protein designs to serve multiple functions, exploiting a biochemical phenomenon called “promiscuity.”

Crenshaw’s focus is on the largest class of natural products, called terpenoids, which are responsible for plant defenses and a number of other functions, including generating aromas that attract bees or deter plant-eating organisms. Oranges, for instance, produce a terpenoid called valencene that smells like…well, oranges. Terpenoids also give mint and lemongrass their distinctive scents. Small changes in the genes responsible for a subclass of terpenoids, known as terpene synthases, allow these enzymes to output diverse chemicals that protect the plant against a range of different foes, including bacteria, fungi and insects. Crenshaw hopes to shed light on the origins, evolutionary history and driving forces responsible for this vast diversification of function.

Tatyana-Sharpee

Tatyana Sharpee

Crenshaw and Sharpee study a terpene synthase from tobacco called 5-epi-aristolochene synthase (TEAS) that helps generate a natural antibiotic, capsidiol, to protect the plant from mold during wet periods. The plant generates TEAS based on instructions in its DNA, and variations in that gene among different plants result in terpene synthases with different shapes. The plants produce versions of TEAS that best help them defend themselves in their local environment. In other words, plants have learned how to combat antibiotic resistance long before we humans even knew antibiotics existed. In the laboratory, Crenshaw and colleagues generated 500 versions of TEAS by mixing and matching mutations that were identified in nature much like others have identified specific mutations in various human cancers. Tweaking the DNA code altered the sequence of amino acids, which in turn changed the enzyme’s properties when the long chain of amino acids folds into a threedimensional structure. When Crenshaw tested the heat tolerance of the different TEAS mutants the results were surprising.

Working with Sharpee and University of California, San Diego graduate student Jonatan Aljadeff to apply theoretical models borrowed from physics to analyze the data, Crenshaw found that many combinations of the mutations she introduced had modest effects on the thermal stability of the molecules. For instance, most of the proteins were stable up to 40 degrees Celsius (or 104 Fahrenheit), within some range. But this wasn’t always the case. Some of her tiny tweaks led to huge shifts in thermal stability, with some of the proteins capable of withstanding temperatures of up to 53 degrees Celsius (127 Fahrenheit).

“Most variations of TEAS have similar characteristics, so it’s almost as if there is a mechanism keeping the thermal stability of the protein close to the status quo,” she says. “But then you’ve got these occasional non-linear effects that pop up, where the proteins are stable at temperatures way outside of the norm. So, resilience to even higher temperatures can be accessed through relatively few DNA sequence mutations. And this is possible without plants losing their ability to synthesize critical natural chemicals used for protection and improved growth.”

“I’m really interested in exploring what’s possible,” Crenshaw adds. “The potential in the plant genome to adapt to wide variations in climate is vast. I believe this is the power and promise of understanding plant evolution to help us adapt to changing climates across the globe.”

Some Like it Hot

Crenshaw’s quest to plumb the possible is really an extension of something humans have been doing for thousands of years. Because we have always relied on plants for our survival, it’s a good bet that we humans have eaten (or gingerly tasted) every plant we’ve encountered. Many of those we liked, we’ve modified to our tastes. In the case of the wild cabbage, for instance, humans selecting for different desirable characteristics turned a single species, Brassica oleracea, into a smorgasbord of crops that includes broccoli, cauliflower, kale and Brussels sprouts. Corn is another prime example. Corn has long been a food staple in the Mexican culture of the Baja California region near the Salk Institute. The modern corn used by Mexican families to make tortillas and tamales is a far cry from the primitive maize plant, which produced a single, spindly cob covered in tiny kernels. First domesticated 10,000 years ago by ancient farmers, millennia of artificial selection have produced the robust, completely domesticated varieties that produce multiple large cobs per plant–each covered in juicy, pea-sized kernels.

In other cases, plants we use for food didn’t need any coaxing to survive–even when we moved into less than hospitable climates. Those Mexican families mentioned earlier also eat wild prickly pear cactus. This thorny emblem of the desert is a great example of a plant that can survive in an extreme environment. In addition to being dry, deserts are also extremely hot and sunny, and as a result cacti and other desert plants are fine-tuned to these conditions. These adaptations are the result of millions of years of natural selection, but what if domesticated crop varieties could be conferred similar properties through traditional breeding or genetic engineering? If so, it might improve crop yield during hot, dry climate swings or allow crops to be introduced to new regions–for example, wheat could be grown in a more arid climate than is now possible. The first step to answering the question is to untangle the molecular pathways and genes that are activated when a plant is stressed by heat and high light–exactly the challenge that Joanne Chory’s team is currently tackling.

In the cells of a plant’s leaves, organelles known as chloroplasts are responsible for photosynthesis, the conversion of light energy into chemical energy necessary for the plant to survive. The energy captured is used to convert carbon dioxide from the air into highenergy compounds like glucose and starch. One of the byproducts of photosynthesis is oxygen, which is released as a waste product and creates the atmosphere we oxygen-breathing creatures require to roam the Earth.

Because chloroplasts are so essential to a plant’s survival, it is critical for a cell to be informed about their functional state and for the cell’s nucleus, its control center, to operate in concert with the chloroplasts. The nucleus contains the majority of the plant’s DNA, but the chloroplasts also contain a short strand of DNA. Chloroplasts were once independent microorganisms, but somewhere along the line were incorporated into plant cells. Chloroplasts contain about 117 genes, 87 of which are code for proteins involved in directing chloroplast function.

Arabidopsis seedlings growing in a petri dish.

In previous groundbreaking research with Arabidopsis, Chory’s team discovered how the nucleus and chloroplasts communicate about the production of the photosynthesis machinery. Parts of the system are built in the chloroplast and other parts are built in the nucleus, but the final molecular complexes are assembled in the chloroplasts. They found that signals sent from the chloroplasts to the nucleus helped coordinate this complex process. In another study, Chory and her collaborators identified a gene that is a key player in signaling between the chloroplasts and nucleus. That discovery laid the groundwork for exploring precisely how plants respond to stress, since coordinating cellular responses to protect and repair chloroplasts is critical to a plant’s survival. It also suggested an avenue for augmenting a plant’s resilience to environmental stressors.

More recently, Jesse Woodson, a staff scientist in Chory’s lab, has identified a mechanism in plant leaves that tells cells to recycle chloroplasts damaged by extreme heat, light or other stressors. By removing the damaged chloroplasts, this system theoretically prevents the entire cell from dying.

“Maintaining an existing cell takes less energy than generating an entirely new one, so this could be a damage control strategy for plants,” says Chory.

In another ongoing line of research, Xiaobo Zhao, a postdoctoral researcher in Chory’s lab, found that a certain gene mutation made plants more sensitive to heat stress. He is now exploring whether amplifying the expression of the normal form of the gene might make plants more heat resistant. “One goal is to identify the entire pathway by which plants respond to heat stress,” Zhao says. “There are general stress mechanisms that are triggered by a number of different stressors, whether it’s a cold snap or a hungry insect, but we think we’ve found a gene that is specifically involved in heat stress. We are also hoping to find ways to control a plant’s heat response, so it could grow efficiently in hotter climates, and this gene is a promising lead.”

A Bright Future

On a related front, the Chory lab is searching for a switch that controls plants’ response to light intensity that is especially high. Plants have evolved to Earth’s particular light cycle and to the light conditions found at the longitude and latitude they call home. Farmers often grow crops that originate in climates that are very different from that of their farms. In places like Australia, Africa, Mexico and the southwestern United States crops have to contend with sunlight that is particularly intense–more intense than many plants need or can withstand.

“When there is more light than the plant needs, the excitation energy exceeds the capacity of the photosynthetic apparatus, which results in a buildup in leaves of reactive oxygen molecules such as hydrogen peroxide,” says Jianyan Huang, the postdoctoral researcher leading the study. “These molecules trigger a stress response to the high light conditions. If the plant’s response isn’t adequate, the excess reactive oxygen can damage the plant–for instance, damage to chloroplasts and the loss of chlorophyll results in the leaves turning pale, a phenomenon known as bleaching.”

Chloroplasts (green), the living solar panels within a plant cell, can be damaged by extremes of temperature and light.

One way a plant responds to extreme light conditions is to put the brakes on its photosynthesis system. When reactive oxygen molecules start to build up, the plant cells reduce the size of the light-harvesting complex in chloroplasts to relieve the oxidative stress. It’s akin to removing the solar panels from your roof on an extremely sunny day to prevent damage to your house’s electrical system.

In ongoing experiments, Huang is trying to identify molecules that trigger the high light stress response by activating certain genetic programs in plant cells. She’s screening a collection of 2,000 Arabidopsis transcription factors, proteins that control which genes are turned on or off. She has identified a number of possible candidates from the collection, and she’s currently testing to see whether they are capable of activating genes known to be involved in the high light stress response. Her experiments have turned up some promising results, including identifying several proteins that appear to switch on the high light response. Similar to Zhao’s work, her findings could lead to a way to develop agricultural crops that can be farmed in places where high light periods are a threat.

“Our work in Arabidopsis and other model organism plants sets the stage for identifying the same genes and molecular pathways in crops,” says Chory. She notes, for example, discoveries made in Arabidopsis have been instrumental in the development of tomatoes rich in carotenoids, naturally occurring pigments that give vegetables their yellow, orange, and red hues. In addition to providing color, carotenoids such as beta-carotene, lycopene, lutein and zeaxanthin are important dietary nutrients. Another variety of tomato developed separately contains high levels of anthocyanins, plant pigments that are antioxidants and have been shown to extend life in mice with cancer. Tomatoes high in anthocyanins are more purple than red–owing to the bluish color of the anthocyanin molecules.

Scientists in academic institutions and biotech companies are also working to develop drought-tolerant crops, both through traditional crossbreeding and through genetic engineering. For instance, the Drought Tolerant Maize for Africa project has generated 153 new varieties of drought-tolerant maize. In early field trials, these varieties yielded 30 percent more under drought conditions compared to commercially available varieties under normal rainfall conditions. Another project, organized by the African Agricultural Technology Foundation in Nairobi hopes to have a transgenic variety of drought-resistant maize available for African farmers as soon as 2016.

“At Salk, we’re not in the business of developing new crops, but we find the genes and pathways that are responsible for the stress response and which can be leveraged to improve agriculture,” says Chory. “The more we know about the plants we rely on, the more resilient we humans will be as a species. Change is coming, that much you can be sure of. The big question is how we will respond.”