The Color of Onions : The Whims of Genetics

I've previously posted about what might go into changing the color of onions (the-biologist-is-in.blogspot.com/2013/12/the-color-of-onions.html), but now I've gone and done an experiment. It was an accident, really, but many useful experiments start out as accidents.

We planted out a batch of "red" onion seedlings this last spring. We got them in a trade from someone who had started them. We got a pot of onion threads, and they got a couple squash babies in return. I'd never grown onions before, so I just put them (along with several types of decorative onions, hoping the deer would leave them all alone) into a raised bed that I had recently cleared. I watered the babies a few times when I noticed the soil was dry. I never fertilized or amended the soil. I basically ignored them. In retrospect, this is not the way to get those luxuriant onions you see in the store. Somehow, almost all the plants survived and produced bulbs. Inch-long bulbs, that is.

I pulled each onion as its leaves died down. They got cleaned, dried, and then left alone on the kitchen windowsill. After too many had accumulated, I moved them to a spare drying rack left over from an ongoing tomato-jerky experiment with a food dehydrator. A couple days later, I happened to notice that one of the bulbs was a much darker color than all the others. An early thought was that this was the color of some mold infesting the bulb, but on close examination there didn't seem to be anything wrong with it. It definitely was a darker shade.

I started looking at the color of the collected onions. One stood out as being more red than the others... actually red instead of that purplish color that "red" onions typically are. Another was a rich purple color.

www.braukaiser.com/wiki/index.php?title=An_Overview_of_pH

Since "red" onions are colored by anthocyanins that change their color depending on pH, we can estimate the pH of the cellular structures where the pigment is found. The red bulb approaches a pH of 2, while the purple bulb approaches a pH of 5. If we could drive the pH further to the right by the same interval, we'd get a pH=8 onion that looked blue.

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I was planning to save the color outlier bulbs (red, purple, dark) to grow the following year for seed. Unfortunately, they didn't survive the winter. I was pretty sure they wouldn't survive outside, but I didn't think about how best to get them to survive inside.

I may re-do this initial experiment next year. Onions with unexpected colors would be fun.


References:

• the-biologist-is-in.blogspot.com/2013/12/the-color-of-onions.html
• www.braukaiser.com/wiki/index.php?title=An_Overview_of_pH

Potato Onion and Gene Networks

I've been growing onions in my garden for a couple years. Like most of my garden veggies, they're not exactly the typical sort. A few years back I received a large sample of "potato onion" true seed derived from work by Kelly Winterton. Potato onions are an old-fashioned perennial form of the typical garden onion (Allium cepa). The "potato" in their name is because they're grown by planting some of the previous year's crop, like as is done with potatoes.

Kelly's introduction into potato onion breeding came from a lucky break, when he planted some of his onions in the fall to see if they could overwinter in the ground at his northerly location. The next season, all of those bulbs flowered prolifically. The important thing he did was to save all those seeds an then try growing them the next year, next to his pre-existing potato onion clones. The high diversity and robust growth of his seedlings caught his attention and started a bit of a movement. (For more details of his work, read through his site.)

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Seedling onions, next to seedling Siberian irises.

I'd read about Kelly's work, so when I had the chance to get a batch of seeds from one of his lines, I jumped at the chance. My first year working with them, I just tossed a scattering of seeds into a 4"x4" pot and let the seedlings fight amongst themselves for the rest of that year. (I wanted to select for aggressive growers.) At the end of the year, I separated the survivors and planted them in the main garden to overwinter. (I wanted to select for those that were very cold-hardy too.) In the end, I had six plants from that first batch.

Two had lots of luxuriant leafy growth, while the other four seemed to grow a little while and then stall out. I was kinda sad most of the plants didn't seem to do anything, but I left them alone until the first frosty night. As I was pulling up the plants, I got some surprises. All four of the poorly growing plants had grown bulbs, with three of them perfectly formed (though small). The two that grew dramatically produced lots of divisions, but no bulbs.

One of the rapid-growing plants flowered twice over the season, so I was able to collect a next generation of seed. Since the plant didn't bulb up the way I wanted, I found myself with a puzzle. I had no idea if those new seeds would all grow into plants with the same growth habit and no bulbs, or if the nice bulb shape could be produced by some hidden recessive alleles. I really liked the aggressive and early growth shown by this plant, so I didn't want to discard its seeds either.

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It took a while of searching before I stumbled on to some useful search queries to get what I was looking for. The first useful paper I found (Lee et al., 2013) goes into detail examining a set of six genes in A. cepa that are related to those associated with the control of flowering in the model plant Arabidopsis thaliana. These "Flowering Locus" genes are transcription factors that regulate how plants develop. The paper has a great deal of interesting information about these genes, but the parts I found most interesting were the experiments showing the interactions between the genes. In this sort of case, I like constructing an interaction network to help me understand what is going on.

Basic model from Lee et al.

The basic model they came up with encompasses three specific genes (AcFT1, AcFT2, & AcFT4). AcFT2 is induced by sufficient winter cold and then induces flowering. AcFT1 induces bulbing and AcFT4 inhibits AcFT1. Sufficiently long days inhibit AcFT4. All together, we get the network at right.

The two larger inferences we can make from this network are drawn dashed and in color. AcFT4 inhibits bulbing (by inhibiting AcFT1). Sufficient daylight hours stimulates AcFT1 (by inhibiting AcFT4) and thus promote bulbing.

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There are a couple more interactions in the biology, so we'll add them. When the flowering pathway is activated in typical onions, the bulbing pathway is suppressed. We'll represent this as a negative influence from AcFT2 to AcFT1, though logically the inhibition could manifest further along the bulbing pathway. The Lee et al. paper doesn't mention this, but I feel this is justifiable from my experience growing onions through to flowering. An interesting point mentioned in the paper, but not discussed in detail was that AcFT4 over-expression plants showed no senescence of leaves in the fall (in addition to no bulbing), instead growing vegetatively until being stopped by winter.

Expanded model for onion.

The Lee et al. paper describes how vernalization regulates flowering in a few other species. They don't examine in detail how it is happens in onions, but their review of how it is regulated in Arabidopsis thaliana gives us a good model for how it might work. The variations in the system in different species does highlight how transcription factor networks can easily be rewired to impact development.

The model doesn't clearly indicate the default activities of the genes. AcFT1, AcFT2, AcFT4, and AtFLC are all active by default. Because AcFT1 and AcFT2 are negatively regulated by AcFT4 and AtFLC, they (and the flowering or bulbing downstream development) are initially inactive.

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The authors in Lee et al. describe how flowering is regulated in a few other model plants for comparison. In short, the same genes are used, but they the comparable interaction network between them has different links. Though the genes are highly conserved, how they work together to drive development is not. The upshot of this is that studies of these genes in other plants is of limited utility to understanding the onions that led me down this path.

Mutations to expanded model for onion.

Even in onions there is evidence for significant diversity in how this regulatory network is put together. In Lee et al., the authors hypothesize that leaks may have an overactive FT4 homolog (shown at left as "X1" on the interaction between daylight hours and AcFT4), resulting in the lack of bulbing and leaf senescence seen in the plant.

Some onions have different vernalization requirements to start flowering. An onion could be entirely resistant to cold as an influence in its blooming, as in "X2" of this figure. Blooming in these onions would be triggered by other influences not described in my figures, such as plant size or age.

The third mutation I have in this figure ("X3") breaks the interaction between AcFT2 and AcFT1. This would prevent flowering from interfering with bulb formation and is what seems to be going on in potato onions. When a potato onion blooms, it will form a bulb from a different growth point that is almost as large as if it hadn't bloomed. This is much different than for regular onions, where flowering results in a tiny inedible bulb.

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Though there are as of yet no genetic studies illustrating this hypothetical mutation in potato onions, it would be relatively easy to undertake. Potato onions and regular onions are the same species and easily cross (if both are flowering at the same time). Sequencing a large number of F2 progeny would help make a connection between variations in genetic sequence and the phenotype of flowering inhibition of bulbing. It might even be faster to sequentially modify one onion type with mutations to match the FT genes (and surrounding regulatory regions) of the other onion type.

The second technique would at the very least be helpful in validating any findings from the first technique, since what I have indicated as a single interaction could actually involve several other genes. The consequence of this could be that the two FT genes might show now sequence differences at all between the two onion types.

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What inspired me to start digging into the biology of bulbing/flowing was the hope that I could make some predictions about the genetics of the potato onions I'm growing.

My robustly growing, but non-bulbing, potato onion seedlings appear to mimic what would be expected if the "X2" mutation described above was involved. I collected seeds from the best of these plants in hopes that they might contain a recessive bulbing trait that would appear in segregations in the next generation. Unfortunately, I still don't know. The phenotype could be due to either a dominant or recessive mutation. I'll just have to grow out as many of these seeds as I have room for and find out. Answering this question will take another two years, so stay tuned.

I'll also be growing a large number of seeds from the batch I originally received. I wasn't expecting so much diversity to appear in them, so now I'm really interested in what other trait combinations will turn up.


References:

• Kelly Winterton: sites.google.com/site/kellysgarden/potato-onions
• Potato onion seeds: www.usefulseeds.com/product/potato-onion-true-seed-green-mountain-f2/
• Primary literature:
• Lee et al., 2013: https://www.nature.com/articles/ncomms3884
• Baldwin et al., 2013: https://www.ncbi.nlm.nih.gov/pubmed/24247236
• Lee et al., 2010: https://academic.oup.com/jxb/article/61/9/2247/532346
• Interaction networks: the-biologist-is-in.blogspot.ca/2016/08/interaction-networks.html

Calculations in the Woods

A. tricoccum in local woods.

Wild foods are available most times of the year in Minnesota, but one species that attracts the most interest in spring is Allium tricoccum (known as "Ramps" or "Wild Leeks"). This slow growing plant is a close relative of onions/chives that are routinely available and has a similar flavor, though aficionados will argue it has a flavor all of its own. Ramps are distinct from the commonly available onion types in that it grows broad and flat leaves, in addition to their habit of growing in the moist shade of wooded areas.

Over-harvesting of A. tricoccum has led to the species disappearing from many areas where they used to be common. The plants grow very slowly, taking several years to grow from seed to a mature plant. The plants are also sensitive to physical disruption because their fragile roots grow close to the surface. If all the plants in an area are pulled out (or accidentally killed), then it could be decades before some seeds find their way back and start towards reestablishing a population.

At this time of year, the local foraging groups are filled with people posting pictures of their (often outrageous) harvests as well as people responding with ideas about sustainable practices of harvest. Advice to, "take no more than half" or, "only take 10%" are pretty common. There doesn't seem to be any standard number. I think some mathematical analysis can maybe help clarify what might be a good rule.

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[1] Lets start with a very simple model. We have a population of plants and a whole bunch of people interested in harvesting them.

If everyone harvests 1/2 of the plants...

\(\lim \limits_{n\to\infty} \frac{1}{2}^n = 0\)

...or 1/4 of the plants (thus 3/4 remain after each person harvests)...

\(\lim \limits_{n\to\infty} \frac{3}{4}^n = 0\)

...then the population still dwindles towards extinction.

In this simplified model it doesn't matter what fraction each person takes, the population will always dwindle away towards extinction. This isn't realistic, since we didn't factor in the ability of the plants to reproduce.

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[2] A slightly more complicated (and realistic) model factors in how fast the plant is able to replicate itself. Lets assume a fraction of of the adult plants are able to produce another adult plant each year. This is still a pretty big simplifying (and highly optimistic, since it is quite biologically wrong) assumption, but it's a starting point to work from. Lets start by defining some terms.

\(\begin{array}{cl}
R_y & \text{Population of Ramps in year 'y'.} \\
r_i & \text{Total increase rate per year.} \\
r_h & \text{Total harvest rate per year.} \\
\end{array}\)

The population of next year is calculated from the current year population and the total rate of increase.

\(R_y(1+r_i) = R_{y+1} \)

Then we add in a term for losses due to people harvesting a percentage of the plants.

\(R_y(1+r_i)(1-r_h) = R_{y+1} \)

If we want the population to remain stable over time...

\(R_y = R_{y+1} \)

\(R_y(1+r_i)(1-r_h) = R_{y+1} \)
\((1+r_i)(1-r_h) = \frac{R_{y+1}}{R_y} \)
\((1+r_i)(1-r_h) = 1 \)
\(1-r_h = \frac{1}{1+r_i} \)
\(r_h = 1-\frac{1}{1+r_i} \)

...and we assume a third of the plants produce a second plant each year,

\(r_i = \frac{1}{3}\)

\(r_h = 1-\frac{1}{1+\frac{1}{3}} \)
\(r_h = 1-\frac{1}{\frac{4}{3}} \)
\(r_h = 1-\frac{3}{4} \)
\(r_h = \frac{1}{4} \)

...then a cumulative total of 25% of the plants could be harvested each year. If any more were harvested, then the population would be declining like in our first model.

Remember, this is the cumulative total harvest rate. This could be just one person harvesting Ramps, or it could be several people harvesting separately through the season. If two or more people come across the patch and decide to harvest some, then they would have to harvest less than the 25% we calculated and still have the population remain stable. We have to define some new terms...

\(\begin{array}{cl}
n & \text{Number of people harvesting in a year.} \\
r_{hi} & \text{Harvest rate per individual per year.} \\
\end{array}\)

The relationship between the number of individuals harvesting and the cumulative total harvest rate is pretty simple.

\((1-r_{hi})^n = (1-r_h) \)

\(\begin{array}{c|c}
{n} & {r_{hi} = 1-\sqrt[n]{\frac{3}{4}}} \\
\hline \\
{1} & {r_{hi} = 1-\frac{3}{4}} = 0.25 \\
{2} & {r_{hi} = 1-\sqrt{\frac{3}{4}}} \approx 0.13397 \\
{3} & {r_{hi} = 1-\sqrt[3]{\frac{3}{4}}} \approx 0.09144 \\
{4} & {r_{hi} = 1-\sqrt[4]{\frac{3}{4}}} \approx 0.06940 \\
{5} & {r_{hi} = 1-\sqrt[5]{\frac{3}{4}}} \approx 0.05591 \\
{\vdots} & {\vdots} \\
{10} & {r_{hi} = 1-\sqrt[10]{\frac{3}{4}}} \approx 0.02836 \\
{\vdots} & {\vdots} \\
{100} & {r_{hi} = 1-\sqrt[100]{\frac{3}{4}}} \approx 0.00287 \\
\end{array}\)

The main lesson we can take from this second model is the more people that have access to a patch of Ramps, the smaller the fraction each person can harvest for the population to remain sustainable.

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From link.

[3] Mathematically, a more ideal model would be somewhere between the discrete series function I used above and a set of continuous differential equations expressing the same concepts as well as accounting for stochasticity in the rates. Biologically, a more ideal model would include each life stage shown in the figure at right (encompassing sexual and vegetative reproduction) as well as realistic rates for each step.

It would be a relatively simple task to construct this sort of more detailed model, but properly determining all the rates would require extensive (presumably years-long) fieldwork. Thus, I'll leave this as an exercise for the reader.

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Even though the models we discussed here are incomplete, they are informative. The big lesson is that the harvesting of Ramps from publicly accessible places is a nice example of a tragedy of the commons. There really isn't a harvesting percentage that can be used as a rule of thumb to tell people in the various forums.

If you have a large patch on your own land, then you can probably harvest a decent amount each year and the patch will never be at risk. Our hypothetical model [3] above might be able to tell us precisely how much of a population could be sustainably harvested, but without all the additional information it isn't worth worrying over. You can simply pay attention to how much you harvest and notice if the patch is dwindling or not from year to year. As it is your own patch, which you find valuable, you will adjust your personal harvest rate to allow the patch to prosper.

Is there anything we can encourage foragers to do, aside from simply advising them to leave the plants alone? If you harvest only one leaf from each mature plant (never the last leaf, or from small plants), without disturbing the bulb and roots, then the plants will survive and spread each year. If everyone followed this rule, large patches of Ramps could be maintained in woodlands close to or even within large cities. Convincing people to do this will be a difficult task.


References:

• www.discoverlife.org/nh/tx/Plantae/Monocotyledoneae/Liliaceae/Allium/tricoccum/
• content.ces.ncsu.edu/cultivation-of-ramps-allium-tricoccum-and-a-burdickii
• www.izelplants.com/allium-tricoccum-ramp.html
• gobotany.newenglandwild.org/species/allium/tricoccum/
• en.wikipedia.org/wiki/Tragedy_of_the_commons 

The Color of Onions

A friend passed me a link to an EBay vendor selling the "Romanian Rainbow Onion" seen at right.

He and I have on several occasions talked about biologically plausible mechanisms that might result in interestingly colored plants, so he was curious if I thought this onion was a real thing.

I responded strongly to the true blue color depicted in some of the inner rings in particular, as being evidence of fakery. Blue is a fairly rare color in biology and I was quite certain that there had never been an onion with such a true blue color... let alone the beautiful color gradient depicted.

At this point I remembered that modern search engines let you search using an image as the query. The right half of the image at left was found in a Turkish news article talking about European onion imports. The left half of the image is the original rainbow-onion image (after being scaled/rotated/cropped to match the placement of the unmodified onion).

Unfortunately, it seems this rainbow onion is the figment of someone's imagination and modern image editing software.

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However, there are biologically plausible mechanisms by which such lovely color gradients could be generated.

www.braukaiser.com/wiki/index.php?title=An_Overview_of_pH[1]

The simplest relies on the interaction between pH the common anthocyanin pigment found in onions, cabbage, and many other plants. Onions generally have a pH on the range of 4-6[2], which corresponds to the purplish shades in the anthocyanin:pH-gradient

There are numerous examples in biology where chemical gradients are generated. Such gradients are critical in developmental biology. It is not inconceivable that an onion could at some point be found (or made) that produced a gradient of pH across the many petiole bases which make up the onion bulb. The gradient that would be produced by this mechanism would not result in the color gradient of the rainbow (ROYGBIV) shown in the original image.

If you want to produce the color gradient of the rainbow, you would have to convince the plant to produce yellow pigments (such as carotenoids) at the appropriate stages of the pH gradient. This more complicated color-gradient system is perfectly plausible in a biological sense, but would be much harder for an agronomist to produce.

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It would be far simpler to just make an onion that was entirely blue, by giving it a pH on the range of 8-10. Not only would this be a dramatic color to add to a salad or burger, it would most likely have a very distinct taste. The sharp flavor we experience from onions is due to the presence of sulfuric acid, so by converting the onion to an alkali pH, we would be removing the major flavor component. Without the strong acid component, perhaps more subtle flavors would be revealed.

To breed for a blue onion, you would only have to select the most alkali bulbs each year to produce seeds from. To keep the project from taking a few more centuries than your life will last with modern medicine, I would advise you to find a way to increase the mutation rate of your seeds.