The Other Route to Biofuels

Enlarge Photo

Photo courtesy of iStockphoto

Using genomics and a range of other techniques, Brookhaven researchers found a means to significantly increase oils in plant leaves—which could potentially enhance the energy content of plants as biofuel feedstocks.

Say you want to convert plant fiber—meaning the inedible trunks, stems, and leaves of plants—into biofuels. That's the goal of most basic research on biofuels today, since it's widely understood that only biofuels made from nonfood plant fiber could be produced in sufficient quantities to put a real dent in our national petroleum consumption. Achieving the scientific breakthroughs to make this production cost-effective is the big challenge.

Interestingly, no matter what process you use for conversion, you're faced with a choice between two broad approaches, each with a fundamentally different chemical focus. The first approach focuses on sugars. Plant fiber contains sugars, but in a complex form. The goal of the first approach is to separate these complex sugars from the rest of the plant fiber and then break down these complex sugars into simple sugars, such as glucose. These simple sugars can then be fermented into fuel, by processes broadly akin to brewing beer.

The second approach is quite different and has received perhaps less attention. It focuses on oils. Plants naturally produce oils, but usually not in huge quantities. If you could manage to coax plants to produce significantly more oil in their stems and leaves, you'd have another fuel source. And oils might have certain advantages over sugars—since removing them from the plants might involve simply squeezing the plants, rather than the complex conversion processes typically required to get at plant sugars. These oils in turn might be relatively easily converted into fuel—and also might even have nutritional value as food.

A number of research teams have been working on developing this latter approach, using today's advanced techniques of genomics, but until recently progress has been slow.

Now scientists at the U.S. Department of Energy's Brookhaven National Laboratory have identified the key genes required for oil production and accumulation in plant leaves and other vegetative plant tissues. Enhancing expression of these genes resulted in vastly increased oil content in leaves, the most abundant sources of plant biomass. The research is described in two recent publications in The Plant Journal and Plant Cell.

"If we can transfer this strategy to crop plants being used to generate renewable energy or to feed livestock, it would significantly increase their energy content and nutritional values," said Brookhaven biochemist Changcheng Xu, who led the research. The experiments were carried out in large part by Xu's group members Jilian Fan and Chengshi Yan.

Think about it in the familiar terms of calories: Oil is twice as energy-dense as carbohydrates, which make up the bulk of leaves, stems, and other vegetative plant matter. "If you want to cut calories from your diet, you cut fat and oils. Conversely, if you want to increase the caloric output of your biofuel or feed for livestock, you want more oil," said Xu.

But plants don't normally store much oil in their leaves and other vegetative tissues. In nature, oil storage is the job of seeds, where the energy-dense compounds provide nourishment for developing plant embryos. The idea behind Xu's studies was to find a way to "reprogram" plants to store oil in their more abundant forms of biomass.

The first step was to identify the genes responsible for oil production in vegetative plant tissues. Though oil isn't stored in these tissues, almost all plant cells have the capacity to make oil. But until these studies, the pathway for oil biosynthesis in leaves was unknown.

"Many people assumed it was similar to what happens in seeds, but we tried to look also at different genes and enzymes," said Xu.

“

The genetic boost resulted in oil production and accumulation of 170-fold compared with control plants, to the point where oil accounted for nearly 10 percent of the leaf's dry weight.”

The scientists used a series of genetic tricks to test the effects of overexpressing or disabling genes that enable cells to make certain enzymes involved in oil production. Pumping up the factors that normally increase oil production in seeds had no effect on oil production in leaves, and one of these, when overexpressed in leaves, caused growth and developmental problems in the plants. Altering the expression of a different oil-producing enzyme, however, had dramatic effects on leaf oil production.

"If you knock out (disable) the gene for an enzyme known as PDAT, it doesn't affect oil synthesis in seeds or cause any problems to plants, but it dramatically decreases oil production and accumulation in leaves," Xu said. In contrast, overexpressing the gene for PDAT—that is, getting cells to make more of this enzyme—resulted in a 60-fold increase in leaf oil production.

An important observation was that the excess oil did not mix with cellular membrane lipids, but was found in oil droplets within the leaf cells. These droplets were somewhat similar to those found in seeds, only much, much larger. "It was as if many small oil droplets like those found in seeds had fused together to form huge globules," Xu said.

Bigger droplets may seem better, but they're not, explained Xu. Oil in these oversized droplets is easily broken down by other enzymes in the cells. In seeds, he said, oil droplets are coated with a protein called oleosin, which prevents the droplets from fusing together, keeping them smaller while also protecting the oil inside. What would happen in leaves, the scientists wondered, if they activated the gene for oleosin along with PDAT?

The result: Overexpression of the two genes together resulted in a 130-fold increase in production of leaf oil compared with control plants. This time the oil accumulated in large clusters of tiny oleosin-coated oil droplets.

Next the scientists used radio-labeled carbon (C-14) to decipher the biochemical mechanism by which PDAT increases oil production. They traced the uptake of C-14-labeled acetate into fatty acids, the building blocks of membrane lipids and oils. These studies showed that PDAT drastically increased the rate at which these fatty acids were made.

Then the scientists decided to test the effects of overexpressing the newly identified oil-increasing genes (PDAT and oleosin) in a variant of test plants that already had an elevated rate of fatty acid synthesis. In this case, the genetic boost resulted in even greater oil production and accumulation—170-fold compared with control plants—to the point where oil accounted for nearly 10 percent of the leaf's dry weight.

Enlarge Photo

Photo courtesy of Brookhaven National Laboratory

Increasing oil content in leaves: overexpressing the gene for an enzyme involved in oil production caused plant leaves to accumulate large amounts of oil in large globules (left). When the scientists added a second gene, clusters of smaller, more stable oil droplets formed (right).

"That potentially equals almost twice the oil yield, by weight, that you can get from canola seeds, which right now is one of the highest oil-yielding crops used for food and biodiesel production," said Xu. Burning plant biomass with such energy density to generate electricity would release 30 to 40 percent more energy, and the nutritional value of feed made from such energy-dense biomass would also be greatly enhanced.

"These studies were done in laboratory plants, so we still need to see if this strategy would work in bioenergy or feed crops," said Xu. "And there are challenges in finding ways to extract oil from leaves so it can be converted to biofuels. But our research provides a very promising path to improving the use of plants as a source of feed and feedstocks for producing renewable energy," he said.

Xu is now collaborating with Brookhaven biochemist John Shanklin to explore the potential effect of overexpressing these key genes on oil production in dedicated biomass crops such as sugarcane.

This research was funded by the DOE Office of Science. Images showing the storage of oil in droplets were produced using microscopes housed at Brookhaven's Center for Functional Nanomaterials (CFN), also supported by the Office of Science.

—Karen McNulty Walsh, Brookhaven National Laboratory, kmcnulty@bnl.gov

Funding

Research and Facility (Center for Functional Nanomaterials): DOE Office of Science, Office of Basic Energy Sciences

Publications

Jilian Fan, Chengshi Yan, Xuebin Zhang, and Changcheng Xu, "Dual Role for Phospholipid: Diacylglycerol Acyltransferase: Enhancing Fatty Acid Synthesis and Diverting Fatty Acids from Membrane Lipids to Triacylglycerol in Arabidopsis Leaves," Plant Cell, published online September 27, 2013.

Jilian Fan, Chengshi Yan, and Changcheng Xu, "Phospholipid: diacylglycerol acyltransferase-mediated triacylglycerol biosynthesis is crucial for protection against fatty acid-induced cell death in growing tissues of Arabidopsis," Plant Journal, Accepted Article.

Related Links

Brookhaven National Laboratory, Biosciences Department, Plant Sciences

Center for Functional Nanomaterials, Brookhaven National Laboratory