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Brookhaven researchers identify key genes for significantly increasing oil content in plant leaves
21 October 2013
Scientists at the US Department of Energy’s Brookhaven National Laboratory have identified two key genes required for oil production and accumulation in plant leaves and other vegetative plant tissues.
In separate papers published in the journals The Plant Journal and The Plant Cell, they report that overexpression of these genes resulted in significantly increased oil content in leaves, the most abundant sources of plant biomass. In one test plant, they achieved almost twice the oil yield by weight that can be obtained from canola seeds. The finding that could have important implications for increasing the energy content of plant-based foods and renewable biofuel feedstocks.
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 the research of studies of Brookhaven biochemist Changcheng Xu and his group’s members Jilian Fan and Chengshi Yan 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.—Changcheng Xu
The researchers tested 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 significant effects on leaf oil production.
Knocking out the gene for the enzyme Phospholipid:Diacylglycerol Acyltransferase1 (PDAT1) didn’t affect oil synthesis in seeds or cause any problems to plants, but decreased oil production and accumulation in leaves, Xu said. In contrast, overexpressing the gene for PDAT 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.
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.
By activating the gene for oleosin along with PDAT, the researchers found that 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 identify 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 increased the rate at which these fatty acids were made.
Then the scientists tested 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% of the leaf’s dry weight.
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.—Changcheng Xu
Overexpression of PDAT1 increases leaf TAG accumulation, leading to oil droplet overexpansion through fusion. Ectopic expression of oleosin promotes the clustering of small oil droplets. Coexpression of PDAT1 with oleosin boosts leaf TAG content by up to 6.4% of the dry weight without affecting membrane lipid composition and plant growth. PDAT1 overexpression stimulates fatty acid synthesis (FAS) and increases fatty acid flux toward the prokaryotic glycerolipid pathway. In the trigalactosyldiacylglycerol1-1 mutant, which is defective in eukaryotic thylakoid lipid synthesis, the combined overexpression of PDAT1 with oleosin increases leaf TAG content to 8.6% of the dry weight and total leaf lipid by fourfold.
In the plastidic glycerol-3-phosphate acyltransferase1 mutant, which is defective in the prokaryotic glycerolipid pathway, PDAT1 overexpression enhances TAG content at the expense of thylakoid membrane lipids, leading to defects in chloroplast division and thylakoid biogenesis. Collectively, these results reveal a dual role for PDAT1 in enhancing fatty acid and TAG synthesis in leaves and suggest that increasing FAS is the key to engineering high levels of TAG accumulation in green biomass.—Fan et al. (2013b)
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 (BES).
Jilian Fan, Chengshi Yan, Changcheng Xu (2013a) “Phospholipid:diacylglycerol acyltransferase-mediated triacylglycerol biosynthesis is crucial for protection against fatty acid-induced cell death in growing tissues of Arabidopsis”, Plant J doi: 10.1111/tpj.12343
Jilian Fan, Chengshi Yan, Xuebin Zhang, and Changcheng Xu (2013b) “Dual Role for Phospholipid:Diacylglycerol Acyltransferase: Enhancing Fatty Acid Synthesis and Diverting Fatty Acids from Membrane Lipids to Triacylglycerol in Arabidopsis Leaves”, Plant Cell doi: 10.1105/tpc.113.117358
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