Scientists engineer yellow-seeded camelina with high oil production

Efforts to achieve net zero carbon emissions from transportation fuels are increasing demand for oil produced by non-food crops. These plants use sunlight to fuel the conversion of atmospheric carbon dioxide into oil, which accumulates in the seeds. Crop breeders, interested in selecting plants that produce a lot of oil, look for yellow seeds. In oilseed crops like canola, yellow-seeded varieties generally produce more oil than their brown-seeded counterparts. The reason: The protein responsible for the brown seed color, which plants with yellow seeds lack, also plays a key role in oil production.

Now, plant biochemists at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory, who want to increase the synthesis of plant oils for the sustainable production of biofuels and other bioproducts, have harnessed this knowledge to create a new variety of high yielding oilseeds. In an article which has just appeared in The Journal of Plant Biotechnologythey describe how they used modern genetics tools to produce a yellow-seeded variety of Camelina sativa, a close relative of canola, which accumulates 21.4% more oil than regular camelina.

“If breeders can get a few percent increase in oil production, they consider that significant, because even small increases in yield can lead to large increases in oil production when you plant millions acres,” said Brookhaven Lab biochemist John Shanklin. director of the Laboratory’s biology department and head of its research program on vegetable oils. “Our increase of almost 22% was unexpected and could potentially lead to a dramatic increase in production,” he said.

Simple idea, unusual plant

The idea behind developing this high-yielding camelina variety was simple: mimic what happens in high-yielding, yellow-seeded landraces of canola.

“Breeders had identified plants with more oil, some of which had yellow seeds, and they didn’t really care about the mechanism,” Shanklin said. But once scientists discovered the gene responsible for both the yellow color of seeds and the increased oil content, they discovered a way to potentially increase oil yield in other species.

The gene contains instructions for making a protein known as Transparent Testa 8 (TT8), which controls the production of compounds that, among other things, give seeds their brown color. Importantly, TT8 also inhibits some of the genes involved in oil synthesis.

Xiao-Hong Yu, who led this project, hypothesized that removing TT8 in camelina should release the inhibition of oil synthesis and release carbon that could be channeled into oil production.

Getting rid of a single gene in camelina is very difficult because this plant is unusual among living things. Instead of having two sets of chromosomes, that is, two copies of each gene, it has six.

“This ‘hexaploid’ genome explains why there are no natural varieties of yellow-seeded camelina,” Yu explained. “It would be very unlikely for mutations to arise simultaneously in all six copies of TT8 and completely disrupt its function.”

Gene editing makes its way to oil

Using modern genetics tools, the Brookhaven team managed to eliminate all six copies of TT8. They used gene editing technology known as CRISPR/Cas9 to target specific DNA sequences contained in TT8 genes. They used this technology to split the DNA at these locations and then create mutations that turned off the genes. Yu and the team then performed a series of biochemical and genetic analyzes to monitor the effects of their targeted gene editing.

“The yellow seed phenotype we were looking for was a great visual guide for our search,” said Yu. “It helped us find the seeds we were looking for by screening fewer than 100 plants, among which we identified three independent lineages in which all six genes were disrupted.”

The results: The seed coat color changed from brown to yellow only in plants in which all six copies of the TT8 gene were disrupted. Yellow seeds had lower levels of “flavonoid” compounds and “mucilage” – both normally produced by biochemical pathways controlled by TT8 – than brown seeds from camelina strains with unedited genomes.

Additionally, many genes involved in oil synthesis and production of fatty acids, the building blocks of oil, were expressed at increased levels in the seeds of the CRISPR/Cas9-edited plants. This led to a dramatic increase in oil accumulation. The modified seeds contained another positive surprise, in that protein and starch levels were unchanged.

The targeted mutations in TT8 were inherited in subsequent generations of camelina plants, suggesting that the improvements would be stable and long-lasting.

“Our results demonstrate the potential for creating new camelina lines through genetic modification, in this case by manipulating TT8 to improve oil biosynthesis. Understanding more details about how TT8 and other factors control biochemical pathways can provide additional genetic targets to increase oil yields,” says Shanklin.