Breeding better crops through genetic engineering has been possible for decades, but the use of genetically modified plants has been limited by technical challenges and popular controversies. A new approach potentially solves both of these problems by altering the energy-producing parts of plant cells and then removing the DNA editing tool so that it cannot be inherited by future seeds. The technique was recently demonstrated by proof of concept experiments published in the journal Natural plants by geneticists from the University of Tokyo.
“Now we have a way to specifically modify the genes of chloroplasts and measure their potential to make a good plant,” said associate professor Shin-ichi Arimura, who heads the group that carried out the research.
Chloroplasts, the parts of plant cells that convert carbon dioxide and sunlight into sugar, have their own circular DNA made up of the same ATGC code as the double helix DNA in the cell nucleus. However, chloroplast DNA is conserved and inherited completely separately from nuclear DNA. Each cell can contain multiple chloroplasts, each with many identical copies of chloroplast DNA. The same change must be made to each copy of chloroplast DNA if a change in the genome is to have a noticeable effect that can be inherited by the offspring of the plant.
In the 1990s, experts invented a technique to insert new DNA fragments into chloroplast genomes, but it also inserts additional genetic tags or markers.
The goal of Arimura and his colleagues is to make uniform and hereditary changes only to specific parts of chloroplast DNA without giving up genome editing tools or permanently altering nuclear DNA. They started with an existing tool known as TALEN. The original TALENs use a large protein that recognizes specific short DNA sequences and cuts that DNA with an enzyme. In recent years, other research groups have improved TALEN technology: DNA recognition sequences can be custom and the DNA-cutting enzyme can be replaced by an enzyme that transforms the GC pairs in the DNA code into AT pairs.
These changes from GC to AT are subtle – just changing one point in the DNA code to another, rather than inserting or removing entire genes. However, point mutations can have major effects depending on their location.
The Arimura team combined these improvements from TALEN and added an additional “chloroplast targeting” component, calling their finalized version ptpTALECDs. For each genome change the researchers wanted to make, they had to create a corresponding left and right pair of ptpTALECD in the bacteria. The design process is complicated because the pairs of large TALENs proteins and the targeting signals of chloroplasts must be expressed simultaneously as a single unit of nuclear DNA.
“Building the ptpTALECD has been an extremely laborious process, but we have a very dedicated masters student who has done almost all of the work, Issei Nakazato,” said Arimura. Nakazato is the first author of the research publication.
After designing the DNA sequence of ptpTALECDs, the researchers then inserted it into plants of Arabidopsis thaliana, a species of thale watercress common in research laboratories. UTokyo researchers are confident that after building them, ptpTALECD could be inserted into many crop species, as this part of the process is a simple and standard procedure in agriculture and botany labs.
PtpTALECD enter the nuclei of plants, then cells produce ptpTALECD in the same way they produce any other protein. The chloroplast targeting sequence ensures that the finished ptpTALECD proteins are transported out of the nucleus into the chloroplasts where they are then supposed to edit every chloroplast genome they come across.
These first generation plants are considered to be genetically modified organisms (GMOs) because their nuclear DNA has been permanently modified to contain the sequence ptpTALECD.
When these genetically modified plants reproduce with themselves through selfing or with unmodified (wild-type) plants, the next generation of plants normally inherits nuclear DNA, which means that genes are mixed and matched between them. ova and pollen. Some seeds inherit the ptpTALECD sequence and others do not.
However, plants still inherit their whole, intact chloroplasts through their “mothers”, the ova. So, no matter what nuclear DNA the next generation of plants inherits, if their female mother plant had altered chloroplasts, the next generation will always inherit the altered chloroplasts.
The researchers then search for the offspring to find plants that have not inherited modified nuclear DNA, but have inherited modified chloroplasts. These members of the second generation of plants and any of their future descendants can be considered non-GMO end products because their nuclear DNA does not contain any of the genetic engineering machines of the ptpTALECD.
Legal definitions vary, but generally countries assess either the end product or the process when deciding to label an organism as a GMO. According to the definitions of the final product used in Japan and the United States, plants produced with this technique are not GMOs. However, the same plants are GMOs according to the process-based definitions used in the European Union.
So far, Arimura’s team has proven that their system works by editing three chloroplast genes and observing the expected effects on offspring plants.
“The DNA of the chloroplast encodes less than 1% of the total genetic material of a plant, but it has a very important effect on photosynthesis, and therefore on the health of the plant. Hopefully this method will be useful in basic research and applied agriculture. Said Arimura.
The researchers are optimistic that the fact that none of the genetic engineering tools are inherited by future generations and that the method only makes point mutations will ensure that the method will be used to produce better crops accepted by farmers and consumers. .
Issei Nakazato, Miki Okuno, Hiroshi Yamamoto, Yoshiko Tamura, Takehiko Itoh, Toshiharu Shikanai, Hideki Takanashi, Nobuhiro Tsutsumi, Shin-ichi Arimura. July 1, 2021. Targeted base edition in the plastid genome of Arabidopsis thaliana. Natural plants. DOI: 10.1038 / s41477-021-00954-6
Plant Molecular Genetics Laboratory (Japanese only): http: // park.
Graduate School of Agricultural and Life Sciences: https: /
Associate Professor Shin-ichi Arimura
Laboratory of Molecular Plant Genetics, Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Phone. : + 81-03-5841-5075
Email: [email protected]
Ms. Caitlin Devor
Strategic Public Relations Division, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 133-8654, JAPAN
Phone. : + 81-080-9707-8178
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This research was funded in part by the GAP Fund Program at the University of Tokyo and the Japan Society for the Promotion of Science (grant numbers 20H00417, 16H06279, 19H02927 and 19KK0391).