With the availability of Arctic Apples around the corner,
there are a lot of questions swirling about how the browning is suppressed when
the apple is exposed to air. The
browning process has great implications for the value of fruits and fruit
products. This natural discoloration
leads to less consumption of fruit, as well as substantial economic loss and
food waste.
There have been a lot of misleading articles on the Arctic
Apple, and even some well-intended articles miss the mark. In the interest of clarity, this article
provides deeper explanation into the science behind the browning process, its
historical genetic modifications, and the mechanism of silencing in the Arctic
Apple.
Fruit Browning
The process of browning is mediated by an enzyme called
polyphenol oxidase, or PPO. The enzyme
can take a variety of chemical precursors (such as catechol or 4-metholcatechol
in apples) and convert them to other compounds that result in an unattractive fruit. There are other nutritional composition
changes as well, which generally are negative, yet minor. The biggest problem
is that consumers don’t eat it if it is not perfect, and a few brown spots
might deter a consumer from a fruit product.
The non-browning trait is nothing new. The grapes that are dried down to make golden
raisins lack PPO activity. They arose
from a spontaneous mutation in the Sultana grape variety, a variety used to
make the traditional brown raisins. That
genetic modification happened in the early 1960’s. The silencing in grapes came
from an unknown error caused by sloppy DNA replication, damage from radiation
from the earth, or DNA mutations from the sun’s UV rays. Time would show that the PPO enzyme is not
processed correctly by the mutant grape, leading to less PPO activity.
A scientific team at the small company Okanagan Specialty
Fruits genetically engineered an apple to exhibit decreased PPO activity. How is that done? The process
is known as “gene silencing”.
Central dogma of molecular biology, and gene silencing. A. DNA encodes the information for the cell. When a gene is activated the information is copied to RNA, an unstable intermediate that then is deciphered by the cell to make a protein, in this case the PPO enzyme. B. Scientists can add a genetically engineered copy of a gene in the backwards orientation. When copied to RNA, the strand effectively cancels out the expression of the native gene. C. Other times a gene can be turned on to a high level, and that causes the native gene (and the added gene) to be shut down and RNA intermediates removed. That's what happens in the Arctic Apple.
Molecular Biology 101
DNA is a durable double-stranded molecule tucked away in the
nucleus of the plant cell. The
information in DNA is copied onto a single-stranded, unstable intermediate
called RNA. RNA leaves the safety of the nucleus, carrying the coded
information out into the main compartment of the cell where the information is
used to guide synthesis of a specific protein, in this case the PPO enzyme. That's Panel A in the figure.
Gene Silencing
Gene silencing targets that RNA intermediate. If you destroy it or interfere with it, its
information is lost and the plant cell does not use the information it encodes.
In this case, no PPO enzyme.
Most of the time genetic engineers add a DNA sequence to the
nucleus that produces the target sequence (in this case PPO) in the opposite
direction to that of the native gene (see Panel B in the figure). In
other words, it creates a matching RNA strand that is a complement, like two
sides of a zipper, to the normally single-stranded RNA molecule and its
resident information. Together the plant’s
original RNA, and the introduced backwards RNA form a stable helical duplex
that looks something like the DNA helix.
RNA duplexes are not really common except in very controlled situations,
and when they happen in the cell it triggers a warning system that targets and
degrades the double-stranded RNA. That
system then uses the sequence information in the degraded RNA to further target
the same sequences. It’s shown in the
figure above.
In this case a backwards sequence of a gene is installed via
genetic engineering. It produces backwards RNA sequence that will bind the
native RNA, and trigger suppression of the product. This is called “antisense
suppression”.
It also is possible to add the forward gene sequence, immediately next to the same sequence in reverse. This approach forms RNA folds back on itself to form a double-stranded
RNA “hairpin”. This approach triggers a
strong response because it creates the RNA duplex that the plant interprets as trouble. The plant then targets that sequence.
Both of these methods, antisense and hairpin-mediated suppression, are commonly used in labs all over the world in many different organisms. The same effect also happens spontaneously in nature.
Neither of these methods describes the engineering of the
Arctic Apple.
But What About the
Arctic Apple?
In the Arctic Apple the gene encoding apple PPO is turned on
at an extremely high level. That’s
pretty simple to do—but ironically, the high expression level feeds back and turns
off the same gene! This is panel C of the figure.
It all started in the 1980’s when Dr. Rich Jorgensen’s lab
wanted to make deeper purple petunias.
They turned on one of the genes limiting the production of the purple
pigments at a super high level and the results were shocking. Sometimes the flowers that should have been
the deepest purple were coming out white as snow or with white patterns on purple flowers. They called this “co-suppression” as the added
gene and the native gene were both turned off. A new branch of science exploded!
This is what happens in the Arctic Apple. The gene for PPO (actually four sequences,
one for each member of the PPO family in apple—it has several copies) is turned
on to such a high level that it triggers a mechanism in the plant to constrain
its levels, turning it off.
Yes, you read it right.
It is turned on so high that it fails to make a product.
It sounds counter-intuitive, but think about it this way. Cells have intricate surveillance systems
that scan for large amounts of RNAs that could be interpreted as a viral
attack. The cell then shuts them
down. That might be the best way to
think of it, even though there is technically more to the story.
Conclusion
This is an example of gene silencing, not much unlike what
was used to decrease the papaya ringspot virus in that crop. RNA silencing can be used to limit expression
of unwanted traits—PPO, viruses, colors, even genes that cause susceptibility
to plant disease. The Arctic Apple
offers a product that could bring more apples into the diet and better profits
for growers. I’ve eaten them, and will be happy to buy them once they are
available.
The complete details are presented in their petition to the
regulators. If you have any questions
that you’d like answered, I’m glad to answer them. kfolta at ufl dot edu!
Kevin’s complete record of funding and reimbursement can be
seen at www.kevinfolta.com/transparency . He has never had any financial
interactions with Okanagan Specialty Fruits, but did get a nice box of Arctic
Apples once. Full disclosure- they were delicious.
