Flounder Antifreeze for Plants
Plants like sunshine; plants don't like freezing cold. Here in Alaska, we have lots of both. Summer's long hours of sunshine produce our gigantic vegetables, which could be even bigger if our growing season wasn't so severely limited by freezing temperatures in both spring and fall. Extending the season by just a few days would allow plants to grow significantly bigger.
Summer's length is beyond human control, but maybe a plant's sensitivity to freezing can be adjusted. Some of the so-called cold-blooded animals have their own antifreeze, enabling them to live in below-freezing conditions. Certain arctic fish survive by producing a protein that keeps their body fluids from freezing down to -1.5 degrees centigrade. This antifreeze protein, or AFP, works by binding to ice crystals, preventing them from growing. AFP is produced in the fishes' livers following directions encoded in the DNA of one gene. If we could copy this gene, then insert it into a plant, the plant might produce enough AFP to survive low temperatures.
In 1990, Dr. Fawzy Georges and his staff at the National Research Council of Canada did just that. They built a synthetic version of the AFP gene from an arctic flounder and inserted it into black Mexican sweet corn. This procedure involves several complex steps: synthesizing the gene, constructing a suitable transfer vehicle for the gene, and introducing the vehicle into corn plants.
To synthesize the gene, Georges used an original strategy. DNA is double stranded, and normally both strands must be synthesized. Georges instead synthesized only one strand, using the cell's own machinery to produce the complementary strand. This mimicking of natural cellular reproduction is also less expensive than synthesizing both strands. The Canadian team used chemical enzymes to trick the cells into replicating the gene.
The synthetic AFP gene was then joined with other segments of DNA to form a transfer vehicle, or plasmid. The plasmid also contained more exotic DNA segments. One was a virus that usually infects cauliflower. The viral segment served as a promoter that instructed the corn cells to manufacture AFP continuously. The other, which Georges inserted just after the AFP gene, produces an enzymatic protein called chloramphenicol acetyl transferase, or CAT for short. CAT causes an easily detected chemical reaction, so this insertion is called a reporter gene. Its action provided a kind of proxy, enabling Georges to gauge AFP production in corn cells.
Getting the gene-laden plasmids into the corn cells was the next problem. Separated plant cells, or protoplasts, have membranes that are impermeable to DNA. The scientists turned to electroporation---making holes with electricity. Georges shocked the protoplasts with two jolts of 250 volts each. This killed many of the corn protoplasts, but many of the survivors gained pores just large enough to admit the synthesized plasmids.
The next step was determining if the corn protoplasts had taken up the AFP-producing DNA. That could be checked thanks to the CAT gene; if the cells showed the presence of CAT, then they were also producing AFP. Georges broke open the protoplasts and mixed them with a chemical that turns blue when combined with CAT. The mixture turned blue. The corn protoplasts were cranking out flounder antifreeze.
Not that Georges' work guarantees instant profits for Alaskan corn farmers. Though the experiment was a success as far as it went, the AFP didn't go far enough. It stayed inside the protoplasts, and true freeze protection requires that the protein enter and protect the intercellular spaces. Apparently the AFP was not exported because the gene did not include the proper cell-signalling DNA sequence.
Though the study did not produce freeze-resistant corn, it did prove that plants can produce antifreeze proteins. That's a first step in producing plants that can expand the limits of the far northern growing season. Thanks to genetic engineering, someday Alaska may produce significant argricultural crops.
