Artistic representation of the effect of editing six copies of the TT8 gene in Camelina sativa. Seeds with inactivated TT8 genes (right) show yellow coloring, reduced thickness of their seed coat, and almost 22% more oil accumulation than wild-type seeds (left). Credit: Valérie Lentz/Brookhaven National Laboratory
Scientists increased Camelina sativa oil production by 21.4% by editing the TT8 gene, paving the way for more efficient biofuel crops.
As initiatives to achieve net zero carbon emissions from transportation fuels gain momentum, the need for oil from non-food crops is growing. These crops harness sunlight to transform atmospheric carbon dioxide into oil, which is stored in their seeds. Crop breeders aiming to maximize oil production often favor yellow-seeded plants because these generally produce more oil than brown-seeded varieties in oilseed crops like canola. This is due to a protein that colors seeds brown, absent in yellow-seeded plants, and which also plays a key role in oil production.
Breakthrough in biofuel crop development
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 Biotechnology,Camelina sativa, a close relative of canola, 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.
The Brookhaven Lab research team (left to right): Jin Chai, Jodie Cui, Shreyas Prakash, Xiao-Hong Yu, John Shanklin, Jorg Schwender, Hai Shi and Sanket Anokar. All are members of the Brookhaven Lab biology department; Prakash and Cui are undergraduate students at Cornell University and Stony Brook University, respectively, and are participating in the Undergraduate Science Laboratory Internship Program sponsored by the U.S. Department of Energy. Credit: Jessica Rotkiewicz/Brookhaven National Laboratory
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 increased oil content, they found a way to potentially increase oil yield in other species.
Gene editing for improved oil production
The gene contains the instructions needed to make a protein called Transparent test 8 (TT8), which controls the production of compounds that give, among other things, 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 hits 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 sequences of DNA within the 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 could provide additional genetic targets to increase oil yields,” Shanklin said.
This research was funded by the DOE Office of Science – in part through a project titled “Improving Camelina Oilseed Production with Minimal Nitrogen Fertilization in Sustainable Cropping Systems” led by the Montana State University; the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), a DOE-funded bioenergy research center led by the University of Illinois at Urbana-Champaign; and Brookhaven Lab’s Physical Biosciences Program. Students supported by the Office of Science also contributed to this research. Additionally, the scientists used a confocal microscope at the Center for Functional Nanomaterials (CFN), which operates as a DOE Office of Science user facility at Brookhaven Lab.
