Saturday, January 25, 2014

A look into non-browning Apples or Arctic Apples

The kid eating an apple from a friend's tree
I'm stuck at home with a cold. Urgh... But it's giving me a chance to catch up on my blog. At the request of one of my Venezuelan high school buddies, this blog will be about Arctic Apples or non-browning apples.

The Arctic Apple is not yet on the market. It's currently under approval in Canada and the US. If approved, it will be one of the very first genetically modified fruits to hit the market. It is being made by a small company in British Columbia, Canada, called Okanagan Specialty Fruits, so there has been no involvement of "big Ag" in the development of the Arctic Apple. The technology for the non-browning apples will be patented and owned by the company.

So, why non-browning apples? I thought it was a pretty odd choice and seemed like a cosmetic, "first-world" issue. But according to this article from the New York Times, 60% of consumers were likely to purchase the fruit (however, a separate survey found that 70% of Canucks did not approve of a genetically modified apple). The Arctic Apple's website highlights the fact that a lot more places would use apples if they didn't brown and that a lot of apples are thrown out. When I examine my own shopping habits, I think it's probably true. I never pick up a bruised apple at the grocery store and my toddler doesn't eat brown apples, even though my husband and I do. If you consider that groceries generally display their best produce, it leaves you wondering how many bruised apples were discarded from the farm to the store. A recent piece in NPR highlights that Americans throw away half their food, much of which is still edible. According to Wikipedia, apples are the third most discarded food in the UK, so it may rank highly in the US as well. The Arctic Apple's website also mentions that pre-sliced apples have additives to keep them from browning, which impact flavor. Personally, I can't taste the additives on McDonald's apple slices, but maybe some people can. The company is also banking on the fact that the juice/cider from Arctic Apples is clear, so they will not need anti-browning treatment in the juice/cider production process. But none of this really matters because in the end, this a product being made by a private company, so I'm going to assume they've done all the marketing research before investing millions of dollars in making a genetically modified fruit. After all, I thought the iPad was a stupid idea and now I own one...

Before describing how the Arctic Apple has been genetically modified, we have to understand why apples turn brown (additional reference is here). Apples begin browning when they are injured: cells break down and release two molecules that were previously in different compartments. The joining of these two compounds in the presence of oxygen leads to the formation of a pigmented polymer. The two compounds that are joined are a) polyphenol oxidases (PPO), which are a class of enzymes, and b) phenols. So when you cut, bite or bruise an apple, you damage cells and bring these compounds into contact with one another. Some types of apples brown more quickly than others because they have more PPO. This is the same process that makes bananas brown as well.

The Arctic Apple doesn't have as much of the PPO enzymes, because the transgene that has been added triggers a process called RNA interference. RNA interference is a naturally occurring defense mechanism against viruses that basically hacks up specific RNA. But scientists have been harnessing the same methodology for decades to decrease the amount of RNA for a gene (I actually used it 7-8 years ago during my thesis). If you manage to get rid of the RNA, then the protein doesn't get made. In the Arctic Apple, they added a DNA sequence that triggers RNA interference for all the different PPO enzymes. The RNA gets chopped up, the PPO enzyme never gets made, so the apples never brown. Apparently, the Arctic Apple manages to turn off the PPO enzyme by ~90%. Not too shabby. It's important to note that the sequence that has been added that triggers that RNA interference process is from the apple itself. It isn't from a different species.

A few additional segments of DNA are in the Arctic Apple which are outlined here. The most controversial of these is a gene conferring antibiotic resistance (to kanamyacin). When working with transgenes, these antibiotic resistant markers are needed in the early stages in the lab so that you can figure out which cells picked up the transgene and which ones didn't.

So what are some of the criticisms of the Arctic Apple and are they legitimate? Here's a list of the arguments I found:

1) GMO Arctic Apples Antibiotic Gene Set to Destroy US Apple Exports. I kid you not: that's actually the title of the article. There's so much going on in this article. Let's go through the arguments one by one:
  • Consumers are worried about antibiotic resistance. Understandable. However, apparently, the antibiotic resistance gene is only turned on in leaves and isn't present in the fruit. In fact, just for kicks, I tried to search for recipes that used apple leaves. All of them had apple leaves made out of delicious pastry, so I don't think that anyone will be impacted by this. Regarding the ingestion of the DNA of the antibiotic resistance gene (i.e. not the protein), I've already covered this in a previous post and it's a dead argument.
  • The use of Kanamycin as the marker gene in GMO Arctic Apples means that EU regulators may refuse to allow imports of not just GM Apples from North America but also all North American apples over cross-pollination and gene transfer fears. Whaaaaaaat??? I'm not sure I understand this. First of all, Okanagan Specialty Fruits is only seeking approval in the US and Canada. Not only that, but the European Food Safety Authority has thoroughly investigated the topic of antibiotic resistance through GMOs. Here's the conclusion: "the current state of knowledge indicates that adverse effects on human health and the environment resulting from the transfer of these two antibiotic resistance genes from GM plants to bacteria, associated with use of GM plants, are unlikely." That quote is from 2009. In contrast, the article provides a quote from the EU where they suggest phasing out the use of antibiotic resistant genes in GMOs. Their quote is from 2001.
  • This is another blow to the struggling EU-US apple relations. Apparently apple and pear exports to the EU have declined >70%, according to article. But this is due to "different regulatory standards and requirements for pesticides and food additives". The Arctic Apple tree does not need any different type of pesticide or herbicide, so this argument doesn't really apply (unless someone is going to suggest that a GMO is a "food additive")
2) The Modified Arctic Apple: Bad News No Matter How You Slice It. Man... I wish I could come up with awesome, pun-filled titles like that. It also has a huge list of concerns, so here we go!
  • A new, almost entirely untested genetic modification technology, called RNA interference, or double strand RNA (dsRNA), is responsible for this new food miracle". So, this one is semi-true. RNA-interference is definitely not a untested genetic modification technology. As I previously mentioned, scientists have been using it in labs for decades, and much work has been done in trying to make gene therapy and drugs using the technology. However, the Arctic Apple is the first commercial genetically modified food that I know of that will use this technology, but I'm sure that it will be the first of many (and several are in the works). 
  • Scientists warn that this genetic manipulation poses health risks, as the manipulated RNA gets into our digestive systems and bloodstreams. Well, there's no reference, so I'm not sure what scientists they're referring to. But this concern is along the same lines as the concern regarding the digestion of DNA from GMOs, which has already been covered in a previous post. But for the sake of science fiction, I imagined the scenario where the ingestion of the manipulated RNA got into our system and actually did something. I think that the apocalypse that the article is implying is the possibility that the RNA might trigger the RNAi pathway within our bodies. But the thing is that I couldn't find any reference to the existence of the PPO enzyme in mammals. But I decided to delve deeper into this fantastic universe that the article was postulating and I took the DNA sequence from the PPO enzyme in the apple and tried to see if there was anything similar enough in the human genome (for all the nerdy-geeks, I did a standard BLAT using UCSC's genome browser). Guess what? Nothing. It couldn't identify anything even remotely similar to the PPO enzyme in humans. I checked in the mouse, dolphin, and a few other mammals. I finally found a small region of similarity in the chicken genome (approximately 5% of the PPO gene was similar to a region in the chicken genome, but I couldn't find any evidence that there were any chicken genes there). I'd like to believe that I kicked this argument in the butt. Keep in mind that if all we had to do was to eat the DNA/RNA of a gene to trigger RNA interference in our bodies, wouldn't we be chugging the DNA/RNA for genes to make us skinny, lower cholesterol, regulate blood sugar, etc? Obviously, it doesn't work that way.
  • ... a pesticide-intensive GMO apple, produced through a risky manipulation of RNA, doesn’t deserve a place on our grocery shelves... As previously mentioned, this apple doesn't need special pesticide or herbicide. It could very well be grown using herbicides/pesticides that are approved for organic use.

Cutest/chubbiest hands EVER!!
The final argument that I read about is that by "hiding" the browning of the apple, consumers will not know how old the apple really is. The company argues that by diminishing the browning, it will make mold and bacteria even more visible, which I think makes sense. I think we'd all have to alter our standards for what we consider a fresh apple to be. In my house, we generally throw out a cut apple by the end of the day, but I could see myself cutting a whole batch of apples at the beginning of the week to last throughout the week, if they don't brown. 

The company's website also extols the health benefits of the Arctic Apple. They claim that by suppressing the amount of PPO in the apple, you actually have more anti-oxidants available to you. I'm a bit skeptical of this claim, because the amount of phenols that get oxidized (browned) seems very small compared to the amount that's in the non-browned portion of the apple. If they could do a study to demonstrate this, it would go a lot further in my mind.

Well, there you have it. My conclusion: I'll buy the apples if they don't cost significantly more than regular apples. If they do cost significantly more, I'll probably buy them only for my kid, while he's being picky about his apples. The spouse wants Arctic bananas, and is hoping they get made soon.

Feel free to comment below. 

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Tuesday, January 21, 2014

GMOs resistant to viruses. How does that work?

During a nerdy web-surfing episode, I stumbled upon a database that listed commercial GMOs, the regions where they've been approved, and a description of the genetic modification. Pretty handy. While scrolling through the list, I observed that one of the more common modifications is to confer viral resistance or immunity to the plant. The transgene that is added to the plant is a protein from the virus itself. And then I wondered, "how does that work? How does a protein from the virus give the plant immunity?" I knew that plants didn't have antibodies, but that was about it. During my bachelor's degree, I took quite a few plant courses ("Plants as Human Resources" was really popular), including plant biochem courses in later years to meet the requirements for my biochemistry major. But thinking back on everything I learned, I don't think that the topic of the plant immune system ever came up.

As usual, the interwebs didn't let me down. I found a very confusing 2006 review in Nature entitled "The plant immune system". Plants don't have "mobile defender cells". Despite the fact that Google will try to convince you that these are accessories for your cell phones, the role of these defender cells (which include our white blood cells) is to swoop in and "zap" foreign cells. Instead of this highly effective mobile defense system, plants have an "innate immune system" where proteins on the surface of cells and within them recognize signals from pathogens and microbes. One study found that the Arabidopsis plant turns on over 1100 genes in less than 1 hour when it recognizes bacterial pathogens. Successful pathogens either manage to suppress the plant's immune response or dodge it altogether.

The next paper I read was a fantastic review written in 1999 by one of the scientists who made the first transgenic plant with a virus protein. Before I continue, the spouse-like voice in my head is telling me that I'll need to explain the structure of a virus.

File:TMV structure simple.png
From Wikimedia Commons
Viruses are weird because it's hard to classify them as "living". They need a host cell so that they can replicate and spread, which is why they infect other organisms. They have several components and I'll focus on two of these in this article: the genetic material which is generally RNA, and a protein coat that protects the genetic material. In order to replicate and spread, the virus uses the host cell's machinery to make more of its protein components. Pretty sneaky, eh? Sort of like a thief breaking their way into your house and taking advantage of your lovely plumbing by taking a dump in your bathroom, on top of stealing all your stuff. That last analogy was inspired by my kid, who is currently pointing at his diaper and saying "Poop". I wonder what he needs?

Getting back to plant viruses. And yes... I did change his diaper.

So the review outlines that the first proposal to create a transgenic plant with a virus coat protein was made in 1981, when the author suggested creating plants resistant to the tobacco mosaic virus. The tobacco mosaic virus was the first virus ever discovered, is one of the most thoroughly studied viruses, and infects species other than tobacco (including tomatoes). The study was a joint collaboration between Monsanto and WashU in St Louis. It took several years until a tobacco plant was successfully made that was resistant to the virus, and where the next generation of plants were also resistant to infection by the virus.  Some plants were not resistant altogether, but it took longer for these plants to develop symptoms when compared to controls. The data were published in the journal Science in 1986 and the process was dubbed "coat-protein-mediated resistance" or CP-MR.

To understand how CP-MR works, several experiments were conducted. In the first, they infected the tobacco plants with the genetic material from the virus, meaning that the virus was missing the protective protein coat. They found that the genetically modified plants were more susceptible to infection by the genetic material when compared to control plants. That suggested that there was something in the coat-protein that had been added to the plant that interfered with the infection process early on. As an analogy, think of the virus as the pulp of an orange, as infection as the mess you leave on your counter when you squeeze out the juice, and the protein coat as the rind. In order to squirt the orange juice all over your kitchen counter, you have to remove the rind or at least cut through it. If there's something preventing you from cutting open the orange or removing the rind, then you'll never make a mess on your counter (i.e. infection will never happen). In this experiment, they handed over a pre-peeled orange for your squeezing pleasure, and found that a mess was easily made. So the results suggest that CP-MR doesn't have to do with the "squeezing" or infection process, rather, something prevents the removal of the rind. In the late 80's, several different studies were performed whose results backed up the hypothesis that the coat-protein transgene interferes with viral disassembly.

A few other interesting studies have been performed over the years, in attempts to better understand CP-MR. Some pretty cool experiments were the infection of the transgenic plants with different viruses that were similar to the tobacco virus, but not identical. They found that it's important for the coat proteins on the virus be similar to the coat protein genes in the transgenic plant, but that the sequence of the RNA in the virus doesn't really matter. In thinking about this, I thought it was an important finding because it would imply that the virus could mutate, but that CP-MR would continue working, although it may be to a lesser extent.

So, I like this whole story about CP-MR for many reasons. I think it's a cool, quirky oddity from nature. But I also think that it's a nice success story for the joint funding of research by public and private funds. Of course, the patent for CP-MR belongs to Monsanto and WashU, but apparently they've been working with different global institutions to create disease resistant crops. I have no doubt that Monsanto is making kajillions of dollars off of this, but it has led to the understanding of plant viruses, as well as the creation of some important crops. Some crops that use CP-MR are squash, papaya, and potato, and there are a few others approved in different countries.

Feel free to suggest future topics below.