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Discovery of a new target to increase vegetable oil content – Applications in bioenergy, chemical engineering and nutrition

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Scientists at Brookhaven National Laboratory have discovered a mutation that causes normal Arabidopsis plants (left) to be sterol-deficient (right). They did extensive genetic and biochemical experiments to establish the essential role of sterol in the formation of oil droplets. Credit: Brookhaven National Laboratory

Scientists ID Sterol Essential for Oil Accumulation in Plants

Scientists seeking to unravel the details of how plants produce and store oil have identified a critical new component in the assembly line. They discovered a particular sterol, a molecule bound to cholesterol, which plays a key role in the formation of oil droplets.

“This research greatly expands our understanding of the molecular factors that govern the formation of lipid droplets, which are vital organelles for the storage and metabolism of petroleum in all eukaryotic organisms,” said Changcheng Xu, a biologist at the Brookhaven National Laboratory of the US Department of Energy, which led the study. The conclusions, published in Nature Communication, may suggest new ways of altering the oil content of a variety of plant tissues.

Changcheng Xu

Brookhaven lab biologist Changcheng Xu and his team explored ways to get plants to accumulate more oils in leaves and stems, abundant vegetative tissue that could be harvested for bioenergetic applications. Credit: Brookhaven National Laboratory

The work may be particularly important in informing genetic engineering strategies aimed at increasing the oil content of leaves and stems. These plant tissues typically do not accumulate petroleum, but they could be designed as an abundant source of sustainable oils for making biofuels and other commodities, scientists say.

The results also apply to the build-up of oil in plant seeds, the main place where oils naturally accumulate in plants. These natural reservoirs of vegetable oils provide nutrition to plant embryos and seedlings, as well as animals and humans.

“We have found that a deficiency of a particular type of sterol causes a decrease in the accumulation of oil in the seeds and leaves,” said Xu.

Green light for oil production

Xu and his team have been working for years to increase oil buildup in the leaves and stems of plants.

“Leaves compared to seeds are much more abundant as a possible bioenergetic material,” he noted. “In addition, because the oil from the seeds is used for food, we try to accumulate oil and other basic bioproducts in parts of plants other than seeds, such as leaves and seeds. stems, in order to avoid competition between food and fuel. “

Lipid droplets in leaves and seeds

Droplets glow green: Scientists at the Brookhaven Lab designed Arabidopsis plants to express a green fluorescent protein attached to oleosin, a protein that stabilizes lipid droplets (LD). In the top row, these LDs are easy to spot under a fluorescence microscope in the leaves of control plants (left) but not in the leaves of sterol-deficient mutants (right). The lower row electron microscopic images show an increase in the size of LD but a decrease in the number of LD in the seeds of sterol mutants. Credit: Brookhaven National Laboratory

The team made progress in getting the leaves to accumulate substantial amounts of oil, using the common laboratory plant Arabidopsis.

They have come up with an effective way to track the buildup of oil. Through genetic engineering, they created Arabidopsis plants in which a green fluorescent protein is always attached to a protein called oleosin. Oleosin only accumulates on the surface of lipid droplets. It is part of the membrane surrounding these oil storage compartments in cells to help stabilize them. If a sample of plant tissue (leaf, stem or seed) contains lipid droplets, they emerge as small green dots under a fluorescence microscope.

“We treated our Arabidopsis plants with a mutagen to try and trigger mutations that would increase oil buildup,” said Xu, using the fluorescence technique to identify strains with more green dots and / or bigger.

Ironically, they made their discovery about sterol in a strain of Arabidopsis that hardly accumulated oil.

“The main focus of the ongoing work was to determine which genetic modification caused this dramatic drop in oil buildup,” Xu said. “We thought that looking for this gene could give us new genes / proteins that are important in the formation or accumulation of lipid droplets.”

Indices for interior assembly

On a microscopic scale, scientists know that lipid droplets form in the “endoplasmic reticulum” or ER of cells. It is an internal network of membranes inside cells (not the membrane surrounding the cell) that acts like a kind of factory – assembling and packaging various materials such as proteins and lipids.

Formation of lipid droplets on the endoplasmic reticulum

Formation of oil droplets: Oil (yellow) collects between the layers of the membrane that forms the endoplasmic reticulum (ER) – a network of membranes in the cytoplasm of plant cells. The droplets develop and eventually pinch to suspend in the cytoplasm. But this process only occurs in certain microdomains rich in sterols and oleosins along the membrane. Brookhaven’s team hypothesizes that sterol is essential for the formation of these droplet-forming microdomains. Credit: Brookhaven National Laboratory

Lipid storage droplets form when oil begins to accumulate between the two layers of the ER membrane, but only in certain regions of the ER. Eventually, when there is enough oil, the small pieces of membrane pinch, leaving the oil encapsulated in self-contained compartments.

As the Brookhaven study shows, studying a plant that does not accumulate these lipid droplets may offer clues to the biochemical factors that govern the process and what is unique about the particular ER fields where it occurs. product.

Focus on the gene

To determine which mutation triggered the dramatic drop in oil buildup, Brookhaven’s team used a technique known as positional cloning, a way to search each region of chromosomes to identify a particular gene responsible for a characteristic of interest. The technique restricted the search to a specific region in one of the plant’s chromosomes.

“This region still contains hundreds of candidate genes,” said Xu.

After using whole genome sequencing to look for any mutations in this region, the team identified a gene they suspected to be involved. The gene encodes an enzyme responsible for a biochemical step in the multistep synthesis of sterol, a cholesterol-related molecule found in ER and other cell membranes.

By selectively “removing” the normal (non-mutated) version of this gene, scientists were able to duplicate the effect of the mutation. That is, the plants with the knocked out gene did not accumulate any lipid droplets. In addition, the addition of the non-mutated gene restored the accumulation of oil droplets.

“This experiment clearly proved that sterol plays an essential role in the formation of oil droplets,” said Xu.

But scientists have gone further. They also studied what would happen if they mutated the 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 which, when deficient, results in low oil build-up.

Mutations in the same genes resulted in reduced oil buildup in leaves and seeds. In seeds, where lipid droplets are easier to see, scientists have also conducted quantitative studies of their shapes and sizes.

Taken together, the results provide evidence for a universal role for this particular sterol in the formation of lipid droplets.

“We believe this sterol is vital for the formation of a microdomain in the ER membrane that is involved in the formation of lipid droplets,” said Xu. “Sterol deficiency results in a defect in the formation of such a microdomain.”

Now that they know what happens when these genes are turned off, 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 explore these strategies in future experiments.

Reference: “Sterols are required for the coordinated assembly of lipid droplets in developing seeds” September 22, 2021, Nature Communication.
DOI: 10.1038 / s41467-021-25908-6

This work was funded by the DOE Office of Science (BES).


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