Biofortified Lettuce not a Bitter Pill

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Calcium is the most abundant mineral in the human body, and most of it is found in our bones and teeth. Without this element to form the hard structures of our bodies, life as we know it as mammals would be in peril. We need it for our nerves and muscles to function properly, too. Many people in the United States are deficient in calcium, upwards of 44%, and in the developing world, these numbers are sadly much, much higher. And as we age, we lose more and more calcium from our bones, leading to osteoporosis. Women are particularly at risk for this disease once they go through menopause.

pill-manWhat can we do? Taking calcium in supplement form can be one way to absorb it, but according to the results of a huge multi-year trial of calcium supplements conducted by the Women’s Health Initiative, these provided only modest benefits. Only women over 60 who took the full regimen of calcium supplements (with Vitamin D) had a statistically significant difference from the control group. And this benefit came with a cost – of more kidney stones.

Nutrition research continues to find wrinkles in the vitamin and mineral pill-popping philosophy, often due to the fact that there are many factors that influence the absorption of needed nutrients. In the case of calcium, vitamin D is important to help its absorption, while antinutrients such as phytic acid (aka phytate) and oxalic acid will inhibit its absorption. One particular form, calcium oxalate, is insoluble in water, and will pass right through us, if it doesn’t irritate our tissues on the way through. (It is often shaped like tiny needles that the plants doubtlessly use to defend themselves.)

Can’t we just eat vegetables that are high in calcium? Not necessarily. Broccoli and spinach come to mind, but the calciferous-ness of these two vegetables is often in the oxalate form – enough that people who are predisposed to kidney stones have to limit how much broccoli they eat for this reason. Though a vegetable may be high in calcium, that doesn’t necessarily mean that it will be in a form that is good for you. Or one you can absorb – calcium in soybeans is not very available, and it takes the processing of turning it into tofu to make it a better source. Phytate found in beans can be an impediment to absorption.

And when calcium is added to fortified foods, it often imparts an unpleasant taste. To get around this, my spouse likes to eat those calcium pills disguised in the form of chocolate candy. (I’m sure it’s about the calcium!)

Finally, vegetables that are high in calcium also tend to be more bitter. Great. It makes you want to forget about veggies and just eat cheese. Maybe that’s why I moved to Wisconsin!

No, actually, here’s part of the reason. I believe that a large part of the answer to our our nutrient needs will be found through modifying the plants that we eat through breeding and genetic engineering. The concept of breeding for nutrient content is relatively new, but the concept of breeding for the bioavailability of nutrients is even newer. A nutrient is bioavailable if it is in a form that can be absorbed by our bodies, and in the proper context to speed that absorption. And a plant-based food that has been fortified with a nutrient biologically is called Biofortified. (Hence the name of this blog!)

How could you breed a better source of calcium? The sources of raw material for plant breeders are the many varieties and closely related species of our crops. Breeders will search these plants for traits that they want to breed into varieties that we can grow and eat. It takes many years to do this for a single crop, and the more complex the trait, the harder it is to do.

First, you could search for variation in nutrient content. If an obscure variety of beans is found with high levels, you could breed that trait in relatively easily. On the other hand, say your a smarter breeder and you also look for variations in phytate levels and also breed a low-phytate bean. Finally, if you’re a breeder ahead of your time, you’ll also test out varieties of beans to see how much of the calcium is absorbed. That would be the most informative, but also the hardest of all to do!

Genetic engineering can also help. If you understand the biology of your nutrient in plants, you can try doing it directly. You could find a gene that has an important role in your chosen nutrient (or antinutrient) and try modifying your plant that way. Keep in mind, breeding also modifies the genetics of plants – you just don’t know how you’re modifying it without doing further research.

A little over a year ago, a monumental paper was published on calcium-biofortified carrots. It wasn’t the first paper on biofortified crops, nor was it the first on biofortifying through genetic engineering. What made it so significant was that it was the first to demonstrate through feeding trials that the modification worked – it increased the absorption of calcium dramatically.

What they did is inserted a gene from a plant called Arabidopsis, that produces a calcium “antiporter” protein. Called sCAX1, this protein is found in the membrane that envelops the large, fluid-filled Vacuole in plant cells, as shown above. It is an antiporter because it uses hydrogen ions that want to move out of the vacuole to ‘pump’ Calcium into the vacuole. Theoretically, if you enhanced the activity of these antiporters, you could make the plant store more calcium in its vacuoloes. And that’s what happened.

But they didn’t stop there. Sure, the carrots had more calcium in them, but was it bioavailable? They first fed the carrots to mice, and to track how much calcium the mice absorbed they ‘labeled’ the carrots with a radioisotope of calcium, 45Ca. By comparing the proportions of 45Ca in the bones of mice fed the biofortified and control carrots, they found that the mice fed the enhanced carrots absorbed twice as much calcium as the mice fed normal carrots. They would need to eat half as much of the new carrot to get the same amount of calcium, the researchers reported.

There’s more. They continued with a human feeding experiment. By labeling the carrots with a rare, yet stable Calcium isotope known as 42Ca, and injecting the 30 participants with another rare form, 46Ca, the researchers were able to measure how much calcium in their bodies came from the carrots. They found that people who ate the ‘super carrot’ absorbed 41% more calcium than if they ate normal carrots. Success!

osteoporosisThe research was widely promoted, and it was also criticized. The general criticism was that it involved genetic engineering, which carries a fairly significant stigma. But this could instead be considered an asset – another case where GE crops can benefit consumers. Some of the more intractable critics criticized the fact that carrots provide little calcium in the first place, and that the total milligrams of additional calcium absorbed was insignificant compared to our daily dietary needs.

But this criticism missed a major point – this was a proof-in-concept that calcium nutrition could be improved with this technique. And although the carrot would not itself solve the problem, imagine if each vegetable in your salad could be improved this way – it could really add up!

Now, I’m pleased to report, there is a new paper involving this same gene, this time in lettuce, and investigating another important aspect of biofortified crops – how do they taste?

Recall that higher calcium content might affect how food tastes. I’d like to call this the Bitterness Barrier. Although bitterness can be caused by many other compounds in your food, it is possible that if you engineered your lettuce to have more calcium you might make it less tasty.

YuckyHow ironic would that be? People don’t eat enough healthy veggies as it is, and if plant geneticists are going to help people eat healthier – it would have to be done in a way that still tastes good. It would do no good to make healthy lettuce that tasted like endives! So Sunghun Park at Kansas State University wanted to find out if using sCAX1 to enhance calcium levels in lettuce would affect the flavor or not.

They started with a lettuce variety called Black Seeded Simpson – it is a common green leaf lettuce that you see in most grocery stores. They made a genetic construct containing the sCAX1 gene, a promoter, and a marker gene. The marker gene is a gene used to make sure that the genetic construct was successfully inserted into the nucleus of the lettuce cells.

The promoter is a DNA sequence that tells the plant what to do with the gene – when to turn it on and off and how strongly the cells express the gene. A common one that plant geneticists use is the CaMV 35s promoter which comes from a plant virus and tells the plant to turn the gene on full blast. The researchers also wanted to test out another promoter called cdc2a, which comes from a gene involved in cell division cycles.

They inserted both versions of this construct into lettuce plants, and checked to make sure it was there using several methods. They took their successful transformations and tested the level of expression of the sCAX1 gene. Was it working?

On the right is a northern blot – a measure of the amount of messenger RNA (mRNA) produced from the gene. mRNA carries the genetic code from the DNA in the nucleus to a structure called the Ribosome where it gets translated into protein. By measuring the levels of sCAX1 RNA you can find out how much of the gene is being produced. The stronger the band, the more RNA is being produced. As you can see in the image, two of the 35s-sCAX1 plants have strong expression, and the cdc2a-sCAX1 plant also expresses the gene, but a little weaker. The bands in the dark image on the bottom are ribosomal RNA, which are used to make sure that the RNA levels between each plant are comparable. Naturally, a normal plant – the Control – did not express the gene.

The plants all grew normally and looked identical, so set to studying the calcium levels of the plants. Did they have more calcium?

Oh yes, about 27-29% more calcium was in each plant. Just like the carrots as before, lettuce plants with the sCAX1 gene inserted had higher levels of calclium.

To make sure that the trait was stable in each generation, they allowed the plants to go to seed, and submitted the next generation to the same tests. They also tested to see if the levels of other minerals changed. Calcium was the only mineral that increased, and by 25-32% in this generation. Success!

Well, not quite, now they wanted to find out how it tasted. Although I imagine they did a little munching while watering the plants in the greenhouse, they put together a panel of five highly trained professional tasters to carefully analyze the flavor of the lettuce in a blind taste test.

They analyzed all sorts of various aspects of the flavor and texture, from Flavor, to Bitterness, Sweetness, Crispness, Metallic, Celery and Spinach-like flavors, to something called “Tooth-etch.” (This is a measure of the chalkiness of food.) Each of these attributes were rated on a scale of 0 to 15, 15 being the strongest.

Without boring you with a table full of numbers, let me tell you a bit about the results. Overall sweetness for the regular control lettuce got an average value of 1.533, while the biofortified lettuce got 1.489. To see whether this difference is significant, they calculated what is called a p-value. This tells you how likely the difference in results could be due to chance. A high p-value means that the difference between the two averages means that the difference is not statistically significant. A low p-value means that it is unlikely that chance played a major role – and that the difference between your two averages are real. In the case of the average overall sweetness of the control versus the biofortified lettuce, they got a p-value of 0.763. This means that the results are 76.3% likely to be due to chance. In other words – they couldn’t notice a difference in how sweet the lettuces are.

The same went for the other characteristics. The musty/earthy flavor of the control was 1.833 and the biofortified one got 1.644. With a p-value of .166, still not a significant difference. The crispness rating was by far the most identical between the two – 5.367 and 5.344 respectively, had a p-value of 0.949. At a 94.9% likeihood that this miniscule difference in ratings was due to chance – you can safely say that this calcium-biofortified lettuce is just as crisp as it should be. Not a single texture or flavor beat the odds – the lettuce was truly the same as its normal counterpart – almost.

One characteristic, the tooth-etch, was almost significant. Generally, a p-value of at most 0.05 is necessary to suggest that the difference in averages is not due to chance – that it is a real difference. A 5% likelihood of being due to chance is not bad, but you really want something like 1% (p-value=0.01) or lower. If you get a p-value of 0.0001, you’re golden!

The “Tooth-Etch” for the control was 0.233, and the biofortified lettuce got 0.989, with a p-value of 0.057 – slightly higher than the 0.05 cutoff. Either there was no difference and this is a statistical blip, or there could be a very minor difference and the test was not powerful enough to detect it. Maybe there was a wide distribution in the tooth-etch ratings given by the taste-testers? Perhaps there is a slight difference, but they would need more than five expert tasters to pin it down.

It would make sense that if the lettuce has more calcium that it could be a little chalkier, but they didn’t really notice it. Besides, if five discerning palates can’t tell the difference, can any of ours?

They also broke it down to look at several difference transgenic lines, and the only significant differences were that one line was slightly sweeter, and another line had a slightly better umami taste. This could be due to the gene being inserted in different locations (and affecting other genes), or being turned on to different levels in those plants. For all practical purposes biofortifying lettuce does not change the way it tastes – particularly the bitterness. You can have your calcium and eat it too. The Bitterness Barrier has been broken!

The one thing I think was missing from this research was that there was no bioavailability test. Although increasing the calcium content of carrots resulted in more bio-available calcium, that was a different species and a different tissue. Biofortification is a relatively new concept, so what we need in this field are more lines of evidence that it works in many contexts. It is highly likely to work, but a small-scale bioavailability study would be a good idea to verify that the lettuce helps you absorb more calcium.

blackSeededSimpsonLettuceNevertheless, this study shows that not only can we enhance the content of important nutrients through genetic engineering, but we can also precisely verify that we’re maintaining the eating quality of the produce. Parents will still have the same old issues with getting their kids to eat ‘yucky’ veggies, but at least it won’t be any worse than before. Besides, if those veggies can be made to pack a stronger punch, one that is verified through nutritional research, maybe parents might feel like redoubling their efforts to make salads rather than giving up and reaching for Flintstones and other pills that might not do much good.

The research is part of the commercialization process for this lettuce, so it seems we will likely see it come to market in the coming years. I will be interested to see how people respond to it, and whether it helps with nutrition at the same time as helping people to understand that it’s not whether it is modified but how it is that really matters. Would you try an enhanced lettuce if you knew it would help you meet your nutrient needs?

Reference:

Park, S., Elless, M., Park, J., Jenkins, A., Lim, W., Chambers IV, E., & Hirschi, K. (2009). Sensory analysis of calcium-biofortified lettuce Plant Biotechnology Journal, 7 (1), 106-117 DOI: 10.1111/j.1467-7652.2008.00379.x

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Karl earned his Ph.D. in Plant Breeding and Plant Genetics at UW-Madison, with a minor in Life Science Communication. His dissertation was on both the genetics of sweet corn and plant genetics outreach. He currently works as a public research geneticist in Madison, WI. His favorite produce might just be squash.


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8 comments to Biofortified Lettuce not a Bitter Pill

  • Bonnie

    Let’s not forget to explore how people got sufficient calcium before there was genetic engineering. Maybe we could get a clue from ancient diets and lifestyles?

  • Brad

    Very cool. I would much rather have a salad than buy a bottle of “supplements”. Too bad people are so freaked out by what they don’t understand.

    Though it’s a little off the topic of this blog, it’s important to mention that weight bearing exercise is a very important factor in keeping bone density.

  • Kim

    We should think first, did the ancient people get enough calcium? Better nutrition is one reason (not the only reason) that people are taller, live longer, etc. than they used to.

  • Iron-Deficient Woman

    I’m not necessarily opposed to this, but I think that there should be some required labeled. Not because of some paranoia about genetic engineering, but because of potential medical conflicts.

    In my case, I have struggled on and off with iron-deficient anemia for more than a decade. One thing I’ve learned from my docs is that calcium is an iron antagonist, so I can’t have calcium supplements or calcium-rich foods within 2 hours of taking iron supplements.

    Since the high doses of iron I require must be spread out into three doses per day, I need to be really careful about what I consume when. If my carrots or lettuce were suddenly high in calcium, that would be great, but then they’d have to be off the list of things I could take with the supplement.

  • KO

    In the broader concept of human nutrition and consumer behaviour. I’d suggest that people who consume good levels of fruits & vegetables are more likely to practice a healthy balance diet that includes sufficient calcium. Adding bioactive calcium may be more beneficial in food products eaten by people who don’t eat fruit & veg daily.

    Also, what are the physiological and nutritional effects that are removed with the lettuce’s “bittering” agent?

  • Mick Mills

    Vitamin D is required for absorption of calcium in the gut. Research suggests that a level of 32.5 ng/nL (of serum 25(OH)D) is an optimal level. The WHI trial never checked vitamin D levels in the women and only used supplements of 400 IU/d (which would only raise levels by about 4 ng/nL). Other trials have shown that 1000 IU/d vitamin D3 supplementation does have a measurable effect.

  • […] staples in developing countries) this can also be done with genetic engineering. Take a look at my post about enhancing calcium content in carrots and lettuce for an […]

  • […] What’s REALLY interesting about this incident is the actual intended purpose of the crops. And no, these trials have nothing to do with Roundup, and they have little if nothing to do with Monsanto’s less savory practices except tangentially. The trials in question appeared to be of the simple kind – the wheat has been altered in a way that should affect its glycemic index, how rapidly the sugars are absorbed into the bloodstream, and that CSIRO is interested in seeing if it has the desired effect when eaten by human beings. These kinds of studies have been done before, such as on calcium-biofortified carrots as described in this post. […]

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