Scientists investigating how plants produce and accumulate oil have identified a new essential component of the assembly line. They discovered a specific sterol – a molecule related to cholesterol – that plays a key role in the formation of oil droplets.
“This research broadens our understanding of the molecular factors that control the formation of lipid droplets, which are vital organelles for oil storage and metabolism in all eukaryotic organisms,” said Changcheng Xu, a biologist at the US Department of Energy’s Brookhaven National Laboratory who conducts the study directed. The results, published in Nature Communications, could reveal new ways to alter the oil content of a wide variety of plant tissues.
The work can be especially important in educating about genetic engineering strategies aimed at increasing the oil content of leaves and stems. These plant tissues don’t normally accumulate oil, but they could be used as an abundant source of sustainable oils to make biofuels and other raw materials, the scientists say.
The results also apply to the accumulation of oil in plant seeds, the main place where oils naturally accumulate in plants. These natural reservoirs of vegetable oils provide food for plant embryos and seedlings – as well as for animals and humans.
“We found that a lack of a certain type of sterol led to a decrease in oil build-up in seeds and leaves,” said Xu.
Green light for oil production
Xu and his team have been working to increase oil build-up in plant leaves and stems for years.
“Leaves are much more common than seeds as a potential bioenergy material,” he noted. “Since the oil in seeds is used for food, we are also working to enrich oil and other bio-raw materials in non-seed parts of plants, such as leaves and stems, to avoid food-fuel competition.”
The team has made some progress in getting leaves to accumulate significant amounts of oil by using the common laboratory plant Arabidopsis.
They devised a neat way to track oil buildup. Using genetic engineering, they created Arabidopsis plants in which a green fluorescent protein is always bound to a protein called oleosin. Oleosin only accumulates on the surface of lipid droplets. It forms part of the membrane that surrounds these oil storage chambers in the cells to stabilize them. If a sample of plant tissue – leaf, stem, or seed – contains droplets of lipid, they will be seen as small green dots under a fluorescence microscope.
“We treated our Arabidopsis plants with a mutagen to try to create mutations that would increase oil build-up,” said Xu, using the fluorescence technique to identify strains with more and / or larger green spots.
Ironically, they made their discovery of sterol in a strain of Arabidopsis that accumulated almost no oil.
“The main purpose of the current work was to find out what genetic modification caused this dramatic decrease in oil accumulation,” said Xu. “We thought tracking this gene might give us some new genes / proteins that are important in the formation or accumulation of lipid droplets.”
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At the microscopic level, scientists know that lipid droplets form in the “endoplasmic reticulum” or ER of cells. This is an internal network of membranes within the cells (not the membrane that surrounds the cell) that acts like a kind of factory – assembling and packaging different materials like proteins and lipids.
Lipid storage droplets form when oil accumulates between the two layers of the ER membrane, but only in certain regions of the ER. Finally, when there is enough oil, the small pieces of diaphragm are pinched off, leaving the oil trapped in self-contained chambers.
As the Brookhaven study shows, examining a plant that does not accumulate these lipid droplets can provide clues about the biochemical factors that drive the process – and what is unique about the particular ER domains in which it occurs.
Cancel the gene
To find out which mutation caused the dramatic decline in oil accumulation, the Brookhaven team used a technique known as positional cloning – a method of searching each chromosomal region to locate a particular gene responsible for a trait of interest. The technique narrowed the search to a specific region in one of the plant’s chromosomes.
“This region still contains hundreds of candidate genes,” said Xu.
After the team used entire genome sequencing to look for a mutation in that region, the team identified a gene that they suspected was involved. The gene codes for an enzyme that is responsible for a biochemical step in the multi-step synthesis of sterol, a cholesterol-related molecule found in ER and other cell membranes.
By selectively “switching off” the normal (unmutated) version of this gene, the scientists were able to duplicate the effects of the mutation. That is, plants with the knocked out gene did not accumulate lipid droplets. In addition, adding the unmutated gene restored the oil droplet accumulation.
“This experiment provided clear evidence that sterol plays an essential role in the formation of oil droplets,” said Xu.
But the scientists went even further. They also investigated what would happen if they mutated genes for enzymes “upstream” of that particular enzyme in the multistep sterol synthesis pathway. And they measured the sterol levels in these mutants.
The detailed studies allowed them to focus on the specific type of sterol that, when deficient, results in low oil buildup.
Mutations in the same genes resulted in decreased oil build-up in leaves and seeds. In seeds, where lipid droplets are better seen, scientists also conducted quantitative studies of their shape and size.
Together, the results demonstrate the universal role of this particular sterol in lipid droplet formation.
“We believe this sterol is critical to the formation of a microdomain in the ER membrane that is involved in the formation of lipid droplets,” said Xu. “The lack of sterol leads to a defect in the formation of such a microdomain.”
Now that they know what happens when these genes are turned off, the scientists suggest that strategies to turn them on and increase their expression could be a way to increase oil buildup in leaves, stems, or seeds.
The team will investigate these strategies in future experiments.
This work was funded by the DOE Office of Science (BES).
