The Color of Beans 6

My ongoing project to produce distinctly blue dry beans occasionally throws a surprise at me. This spring, two plants with bright red flowers appeared in one of my gardens. The blue lines had until now showed a mix of white and pale pink flowers, so this change was rather dramatic.

I immediately began thinking about how these plants came to be. They clearly grew from blue seeds I'd planted, but that was all I knew initially. Last year I had some (more or less) intentional hybrids turn up in my blue bush bean patch, so I thought they might have represented back crosses to the parental blue line.

In another garden this year is filled with an F2 population from that intentional hybrid I mentioned. If the red flower color came from a back cross, then the same color should also turn up among the F2 plants. Only white and pale pink flowers were evident when they started blooming, thus the red flower did not come from a back cross.

I did have one red-flowered Phaseolus coccineus (var "Insuk's Wang Kong") growing last year. The seed germinated late and the vine never prospered. It had only one small flower cluster and produced a single pod with a single seed. It was also on the far end of the garden from the blue bush population, with rows of large tomato and other bean plants in between. I didn't expect there would be any chance for pollen transfer like this, even though I was hoping there might be some crosses with more nearby plants because I'd been wanting to work with a P. vulgaris x P. coccineus hybrid for years.

I was eagerly waiting for the first seed pods from the red flowered plants to mature, as their seed color would likely be the definitive evidence for the plants being hybrids. The first pods broke open to reveal tan seeds marked with dark blue. The speckled trait came from the P. coccineus parent and the blue color came from the P. vulgaris parent (the blue seeds here for comparison purposes).

Hybrids between P. vulgaris and P. coccinues are often described as having growth issues or have poor seed-set, though the literature and experience of others seems to be quite variable. Fortunately, my hybrids show no such issues but perhaps they may show up in the next generations.

---

Because the P. coccineus parent's seeds were a pale purple, I was expecting the hybrid to turn up with a distinctly purple color and I wouldn't see the blue again until the next generation. Since the speckled trait seems to be dominant, I would expect 75% of the next generation to also show speckles. I can't really predict what the colors in the next generation will be, since I don't have a good idea of what the mixed up genetics will do.

I have read from a few references that the P. coccineus chromosomes are preferentially lost vs the P. vulgaris chromosomes in subsequent generations, so any predictable genetics ratios are likely to be distorted significantly. I'll just have to find out. Because of this tendency, similar crosses have been used before to introgress traits into P. vulgaris, such as disease resistance factors. I'm hoping I can stabilize the flower color over the next few years, as well as the tendency to bloom very well.

References:

1. Interspecific hybridization between cultivated american species of the genus Phaseolus.
• https://link.springer.com/article/10.1007/BF01902923
2. Embryo development in reciprocal cross of Phaseolus vulgaris L. and P. coccineus Lam.
• https://pubmed.ncbi.nlm.nih.gov/24270536/

Chile breeding

Plant breeding involves a great deal of luck and you can have some really good success with that. Luther Burbank produced numerous amazing plant varieties, but at the same time, he never believed in genetics. The key aspects of his method were to just try things, to grow large numbers at every stage, and to pay close attention to every plant (to find the differences). He tried crossing a strawberry and a raspberry... and actually got something.

With some knowledge, you can make predictions about what is more likely to work. To that end, plant breeders have researched what techniques are needed to hybridize different species. Usually there is limited success with hybridizing species in different genera, where the plants are less closely related, but there are exceptions. In the end, species labels are things we make for our convenience. Nature is under no obligation to follow what we think a species is or should be.

---

In peppers there are several domesticated or partially domesticated species:

• Capsicum annum : common sweet & hot peppers in North America.
• C. baccatum : more common in South America.
• C. chinense : habanero type peppers.
• C. frutescens : generally smaller, less common peppers. 
• C. pubescens : rocoto, manzano, locoto; tropical peppers with black seeds.

There are many more wild species that are only occasionally or rarely grown by gardeners. Those species contain a wealth of potentially useful genes though. Disease resistances, agronomic traits, and novel flavors can all potentially be moved from the crop wild-relatives into domesticated peppers. Towards that end, researchers have tried crossing almost any pair of species related to domesticated peppers. The following figure shows a crossability polygon, summarizing the results from attempts to hybridize between eleven species in the pepper genus.

I found the figure several years ago at: www.plantsciences.ucdavis.edu/vc221/pepper/PEPPERrd.htm. I have not yet been able to find the paper in which this figure was first published.

There are other wild pepper species, which could maybe be included in a more recent version of the figure.

• C. lanceolatum: a cloud-forest species sharing the trait of black seeds with C. pubescens.
  C. rhomboideum: a species barely considered to belong to the genus Capsicum, with yellow flowers, sweet berries, and brown seeds.
• C. annum var glabriusculum : tiny, very hot, wild pepper of the desert southwest US.
• And many others...

---

What this diagram suggests is that you could transfer an interesting trait from any one of these species into any other species you're interested in working with, either directly or through intermediaries. It would just take motivation, time, and money to do so. More generally, hybrids between the species would let one introduce much more genetic diversity into their pepper breeding projects, even if there wasn't a specific trait of interest.

I'm working with lines that include C. annum var glabriusculum in their recent ancestry. This was the wild pepper which grew in San Antonio and Austin Texas where I grew up. I started by growing seeds for the species which I (or friends and family) had collected from the wild. At one point, a few seeds collected from a large, seemingly wild plant grew up showing the parent plant had been a hybrid with some unknown domesticated type. I've been growing the descendants of those plants since.

At left is an side view of a patch of miniature pepper plants grown in 2022. Here they're about 4.5 inches tall and the tallest matured to about 8 inches in height. The smallest stayed under an inch (and didn't mature any pods). This line has the small leaves and pods of its wild ancestor, but has a far more dense branching structure and short stems. They produced more and more dark purple/black pigment in the leaves as the season went on. The pods matured from black to bright red at final maturity. The pods range from very hot to volcanic. The plants were covered in black and red pods at the end of the season.

Other plants derived from that initial wild hybrid had larger, more typical forms, though they were still small in size compared to many garden types. Among those, one plant (at right) stood out as extra productive in 2021. I had been hoping to select up a productive hot pepper type since the more standard types have been not doing so well with my short growing season. I grew 25 plants in 2022 from seeds saved from this plant.

Those plants made it clear the productive parent plant had been a new hybrid, between the wild hybrid derived plants I was growing and the Pimenta da Neyde pepper I had been growing the year before. In addition to a wild range of leaf color and plant growth habit in these F2 plants, I also found several with decently large pods at various heat levels.

The split pod at left came from a single plant which seemed to mimic a habanero. The pods had very thin walls and had a heat comparable or higher than habaneros. The internal membranes look so enriched in capsaicin oils that I was hesitant to try tasting them. Other plants seemed to mimic jalapenos, with their thick flesh and corked skin. These peppers are smaller than real jalapenos with a moderate heat level. The plants with both types of pods were far more productive than any habanero or jalapeno I had grown in my gardens, so I took this as a win.

Though I can't be sure, I have a pretty good feeling that all of these peppers are able to grow as well as they do for me in part because of some genes inherited from their recent wild ancestor. They handle the poor soil and limited water in my garden far better than usual garden types. The miniature peppers absolutely have their size and growth habit in part due to their wild ancestry.

In the next several years I hope to stabilize several new varieties from this project. Even if I don't, I'll be getting plenty of hot peppers along the way.

References:

• Burbank's strawberry/raspberry hybrid: https://the-biologist-is-in.blogspot.com/2015/01/hybrid-sterility-and-speciation.html
• Intergeneric hybrid orchids: https://www.aos.org/orchids/additional-resources/intergeneric-hybrids.aspx

The Color of Beans 5

After several years of looking for a truly blue dry bean, I started actively working to produce my own variety in 2018 when I was gifted a few bluish-black hybrid seeds from a bean collector. At the end of 2022, I was essentially done. Five years of selecting the best blue seeds each season and my new variety finally produced a consistently blue crop.

There's probably still going to be a few more years of increasing the seed before I'm prepared to distribute them in some fashion. Along the way I'll be selecting for increased productivity and having to figure out where I can grow more of them each year.

Since harvesting the most recent crop, I've been digging into what research literature there is about the genetics and biology of color in beans. This was initially just to get a better idea of what was going on in my beans. Along the way I realized I could probably use the knowledge I had gained to intentionally make new blue varieties.

---

The initial random hybrid was between a black variety (Haudenosaunee Skunk) and a yellow-tan variety (Arikara Yellow). That color combination would clearly work as parents in a new cross, but I realized there was a combination of parental color genetics that would probably make it easier to recover the right combination of genes to produce a blue color.

The color of black beans is caused by a combination of red, blue, and yellow-brown pigments at high intensity. In Phaseolus vulgaris, a small number of genes lead to this color: R ("red"; likely a transcription factor driving enzyme F3'H), V ("violet"; enzyme F3'5'H), and then B ("brown"; transcription factors enhancing CHS/CHI, darkening all downstream pigments). A yellow-brown bean is caused by all three genes being inactive (written as "rvb"). There are a few other genes that seem to impact exactly what shade of brown color may result, but there doesn't seem to be as much clarity in the research literature about that part of the pigment pathway.

The difference between a purple and a black bean is that the purple bean has an inactive version of B (b). Crossing a purple bean (RVb) to a yellow bean (rvb), where both have the recessive inactive b allele, reduces the genetic possibilities in the F2s to only those impacting the red and blue pigments. Among the F2 plants, we should see the ratios of a dihybrid cross play out. 9 purples, 3 reds, 3 blues, and 1 yellow-brown. 3/16 isn't that bad and I can easily grow enough plants to expect to be able to find the blues I'm looking for.

To test this model I've been building, I decided maybe I could try to make a blue version of one of the other domesticated bean species. Most P. coccineus (runner or ayacote) beans I'd ever seen before were lilac and heavily spotted with black. After digging around for a while, I eventually ordered some mixed color packages from some Mexican bean importers in the south-west. Among those, I was able to select out seeds with the colors I was looking for.

While I was waiting for those to arrive, I was also looking for P. lunatus (lima) beans with the same colors. The yellow-brown variety I found is called Pima Orange. I ordered the purple variety from someone on Ebay and am still waiting for them to arrive.

I wasn't able to find any sign of a purple version of P. acutifolius (tepary) beans. I did find black and yellow versions that I could use with more difficulty. I'll keep looking for a purple version and may eventually decide to try using a black variety instead, but for now I'll hold off on trying to make a blue variety of this species.

---

The color of a seed is determined by the genes of the mother plant, so it can take some tricks to sort things out. The plan will take a few years to play out.

• 2023 : Plant purple & yellow seeds on a common trellis. Some harvested seeds may be hybrids, but we can't identify them yet. Save them separated by color.
• 2024 : Plant yellow seeds only. Save harvested seeds by color.
• Yellow seeds : mother plant wasn't hybrid.
• Purple seeds : mother plant was a hybrid between purple & yellow plants. These seeds will grow into F2 plants.
• 2025 : Plant purple seeds only. Save harvested seeds by color.
• The F2 plants should fall into four categories (9 purple : 3 red : 3 blue : 1 yellow). I've already determined I can grow F2 plants in a mass planting and observe the expected ratios in the cumulative produced seed counts.

P. coccineus and P. lunatus are strong out-crossers, so I can rely on bees and other pollinators to do the work of transferring pollen for me. The two species can't cross, so I can do both parallel experiments in a relatively small garden space. To increase the odds of the yellow seeds produced in 2023 being hybrids, I can plant many more purple seeds than yellow (or even only one yellow seed among many purple). This will result in most flowers that a yellow-seed plant can cross with being those from purple-seed plants.

If no purple seeds turn up in 2024, then no useful cross-pollinations happened in 2023. In 2025 I would then plant purple and yellow seeds to try and find crosses again. There may be some hybrid seeds among the purples, but distinguishing them from the non-hybrids would take a couple more years and require individual plants to be grown on separate trellises. That's more work than I want to put into it, hence designing the plan the way I have.

---

This plan assumes the genes driving F3'H (red anthocyanins) and F3'5'H (blue anthocyanins) are unlinked in these species. I know in P. vulgaris the two genes are not tightly linked. If they were, I would not expect to have been able to find the initial blue-seeded plant so easily as I did. That it is possible to make hybrids between P. vulgaris and P. coccineus tells us their genomes are organized in largely the same fashion, so the two genes should be similarly not tightly linked in P. coccineus. Making hybrids with P. lunatus is harder, but still possible, so similarly I don't expect the two genes to be tightly linked.

Why aren't there already blue varieties of these species available? I don't know, but I feel it might be the same reason that blue varieties of P. vulgaris are so very rare, whatever that is. That and the vast majority of people who have ever grown beans have not been geneticists backed up with decades of published research into the biology of bean pigments. In a few years, I hope to have remedied this absence.

References:

1. Bean varieties:
1. Haudenosaunee Skunk: https://exchange.seedsavers.org/page/variety/id/193592
2. Arikara Yellow: https://www.seedsavers.org/arikara-yellow-bean
3. Pima Orange : https://www.nativeseeds.org/products/pl011
4. Black Tepary : https://www.nativeseeds.org/collections/tepary-beans/products/pt082
5. S'oam Baw Tepary : https://www.nativeseeds.org/collections/tepary-beans/products/pt120
2. Blog posts:
1. https://the-biologist-is-in.blogspot.com/2018/10/the-color-of-beans-1.html
2. https://the-biologist-is-in.blogspot.com/2022/12/the-color-of-beans-2.html
3. https://the-biologist-is-in.blogspot.com/2023/01/the-color-of-beans-3.html
4. https://the-biologist-is-in.blogspot.com/2023/02/the-color-of-beans-4.html
3. Bean species hybrds:
1. https://link.springer.com/article/10.1007/BF01902923
2. https://www.semanticscholar.org/paper/Hybrid-plant-of-Phaseolus-vulgaris-L.-and-P.-L.-by-Kuboyama-Shintaku/c20048408e38632b8ae8fe45d234e967ad57df2d

The Color of Beans 4

In my last post (https://the-biologist-is-in.blogspot.com/2023/01/the-color-of-beans-3.html), I shared a couple figures illustrating the flavonoid/anthocyanin pigment pathway in plants and in common beans (Phaseolus vulgaris) specifically. A couple days later, I found some additional evidence which led me to feel the need to update my figures somewhat.

---

Certain combinations of published genes lead to production of a brown pigment when an excess of yellow astragalin would be expected. Here I've made up an enzyme called FNR (FlavoNol Reductase) leading to production of the pigment, modeled after the production of proanthocyanidins by ANR (Anthocyanin Reductase).

The evidence for a brown pigment derived from the yellow pigment pathway comes from the gene B changing a bean color from yellow-brown to mineral/dark-brown when present. B is thought to be a transcription factor that enhances the expression of other pathway genes. Yellow can only arise when red and blue pigment branches are absent, so the brown pigment produced with B can't be the brown proanthocyanidins derived from the red and blue pigment branches. Instead it must be an analogous pigment produced from kaempferol and/or astragalin. I haven't found any research papers discussing this pigment pathway branch, but all the evidence seems to point to it being there anyhow.

The figure trimmed to the metabolites and pigments significant in common beans... now includes pelargonidins. This version of the figure has different thickness arrows in places to illustrate when one branch has a higher priority, when one branch of metabolites is more likely than another.

Dihydrokaempferol is a central metabolite for all the different pigment branches in the pathway. F3'H and F3'5'H leading to red and blue pigments have the strongest branches, followed by FLS leading to yellow pigments, and then last is DFR leading to orange pigments. For the orange pigments to dominate the final color, it looks like the other three branches have to be disabled or significantly reduced.

So. Are there orange varieties of common bean? Maybe. Instagram user @g3netic_lottery shared a mixed variety of beans they grew this year and some of them had a distinctly orange-ish color, especially in the hilium ring. Now, I'm not entirely sure that is the color of pelargonidin, but it is the closest to orange I've seen in common beans. It's enough of a suggestion that I decided to rework the pathway figure to include it for this species.

I still think it would be really cool to find (or breed up) a variety that had distinctly orange color all over the seed. These beans at right hint that it is feasible if one has the right sort of luck. @g3netic_lottery on Instagram grows beans and other crops in South Africa. I'll be keeping my eye out for useful seeds more accessible to me that might help me get to a nicely orange common bean.

---

The other domesticated bean species will have very similar pathways as the common bean, but the specific available mutations are going to be different. Maybe we can find a good pelargonidin orange color in one of them. I spent some time looking around for orange seeded varieties of the other species, with limited luck.

• Runner beans (P. coccineus): This photo of "Ayacote Mexican" beans includes some very orange looking seeds, but I have no idea how much that reflects reality.
• Lima beans (P. lunatus): "Pima Orange" has seeds that look yellow-brown or orange depending on the photo. Again, it is hard to say what they really look like.
• Tepary beans (P. acutifolius) : There are some photos around of very orange tepary beans, but I can't find any varieties available for sale that look anything near orange.
• Year-long beans (P. dumosus) : This species is interesting, but only grown in a limited area. I found several articles about it, but I have yet to find anyone selling seeds.

I ordered the runner and lima bean varieties above, so I can see what their colors actually look like. It is so very easy to intentionally or accidentally tweak the color of a photo, so the color of photos online isn't always something you should trust.

The runner beans turned up first. Unfortunately, there was nothing like orange among them. I did get the lovely dark purple beans at left though, which are perfect for another project I'll describe in another post. After further looking around, I found a different vendor selling ayacote beans that are at least advertised with some nice orange tones. As they too are selling the beans intended for food use, there's no guarantee any yellow seeds will turn up.

The lima beans arrived a few days later. When I opened the package, I was greeted by seeds with a surprisingly nice orange color! Now, this isn't anything like the orange you get from beta-carotene in some carrots and tomatoes, but this might just be what a pelargonidin orange looks like.

Alternately, this could be a mix of a yellow and a brown pigment. There may be some simple basement-lab tests I can do to help me identify the pigment, but that will take some further research.

In either case, I think it is entirely reasonable to expect a similar orange colored common bean should be possible. It's just a matter of finding the right mutations and crossing them into the same plant.

References:

• Some seed sources:
• dualsnatural.com/products/ayocote-mexican-beans : P. coccineus "Ayacote Mexican".
• https://masienda.com/products/ayocote-beans : P. coccineus "Ayacote Pinto".
• www.nativeseeds.org/products/pl011: P. lunatus "Pima Orange"
• www.nativeseeds.org/collections/indigenous-seeds-bean-tepary: P. acutifolius varieties.

Potatoes

Years ago I heard a story. The story teller's cousin has been growing potatoes and tomatoes each of the last several years. Some years, her cousin reported finding hybrid plants that produce edible potatoes and green tomatoes.

From the story, second-hand as it was, I imagined ripe-red tomatoes on a plant that when pulled up had mature potatoes hanging from the roots. There is usually some truth behind a story like this, but perhaps not what the originally teller or the later hearer initially thought.

The two species (tomato, Solanum lysopersicum; potato, S. tuberosum) are in the same genus, but are distantly related enough that they can't cross to form hybrids. Plants with the important traits of both species can be purchased or made at home, but they are the result of actively grafting separate plants together and wouldn't accidentally form in the garden.

---

As potatoes are in the same genus as tomatoes, they do produce fruit which is very similar to those of tomatoes. However, instead of being large and colorful, potato fruit are small and generally remain green. Most people who've grown potatoes have never seen the fruit because many commercial varieties rarely flower and even more rarely set fruit. Commercial varieties have been bred to not flower and/or produce fruit because that would take energy wasted that could otherwise be used to grow tubers.

The fruit are generally considered to be poisonous (due to high solanine content) like the other non-tuber parts of the plant. Solanine can usually be detected as a bitter taste, but sensitivity to it varies and people have been sickened or killed by consuming too much of it.

Professional potato breeders have been actively reducing the level of solanine in potatoes for decades. A side-effect of this selection process could be the reduction of solanine in the berries as well as the tubers, but nobody seems to have been researching this possibility.

The story teller's cousin could have been growing a potato variety that coincidentally had extra-low solanine levels in its fruit, or they could have a low sensitivity to solanine. Either way, they probably wouldn't have been able to collect enough fruit from the potato plants to be at risk of being in any real danger from the amount of solanine.

A weekend prior to when I first started writing this post, I gathered a batch of fruit from some Yukon Gold potato plants. I tasted one and found it mildly bitter. My mother in law tasted it and found it terribly bitter. We had sampled a similar amount of the fruit and so our different reactions likely reflect different abilities to taste the solanine.

---

I wouldn't advise eating the tomato-like berries to be found occasionally on potato plants, but they have other uses.

Potatoes are typically grown from seed potatoes (either saved from the previous year or bought anew from tissue-culture labs) and thus are genetic clones that will grow/produce very consistently from year to year. The true potato seeds (TPS), however, are the result of a cross- or self-pollination. The mixing up of the parents' genetics means every plant grown from true seed will be different.

As the only potatoes growing in the patch were Yukon Gold, the seeds in the gathered fruit likely represent the result of a self-pollination. Every plant that grows from TPS is instantly a new variety that can then be cloned by saving the tubers. Over a few years one could grow a significant number of new varieties from any given cross, in time developing an appreciation for the genetics that are found in the parent(s). Yukon Gold is a popular variety and others have already performed exactly this experiment. 

A forum discussion about TPS about the likely results of growing seeds from Yukon Gold, written by Tom Wagner, a notable tomato and potato breeder:

I have been testing Yukon Gold OP berries for 25 years or more ever since the experimental clone was first accessed by me. In controlled self pollinated berries, as opposed to OP berries, I get a rather predictable segregation of types each time I grow out seedlings. If you grow out seedlings yourself enjoy the following:

• whites with white flesh
• whites with light yellow flesh
• yellows with light yellow flesh
• yellows with medium yellow flesh
• yellows with deeper yellow flesh
• repeat of above but with either light pink eyes/red eye
• all of the above with templates of size, yield, shape, flavor, etc., differences.

Yukon Gold was selected from a cross between Norgleam (female) and W5279-4 (a yellow-fleshed diploid hybrid of S. phureja and haploid cv Katahdin). Yukon Gold is a tetraploid because of the unreduced gamete from it pollen parent.

Yukon Gold is a tetraploid with a complex parentage, potentially giving it a wide range of possible genetic combinations. That most of those possibilities seem to fall into a distinct set of combinations just means that for those visible traits there isn't that much diversity hiding inside the parent. If you want to play with intensely-colored tubers you would have to look elsewhere.

The really interesting thing about growing potatoes from TPS is all the minor variations in the plant that might impact production or how the plants grow. Over a few years one could select a variety perfectly-suited to the peculiar conditions of your garden, rather than hoping that the default clones available in the store will work for you. At least that's the theory, if you've got enough time and space to dedicate to the task.

---

A few years back I ordered some true potato seed from cultivariable.com to start experimenting with in my garden. Most of the seedlings each year have failed for one reason or another.

A few plants didn't produce a single tuber. Most of those that produced tubers got infected with what might have been late-blight (Phytophthora infestans). For my purposes, the specific disease didn't matter. Any with an obvious infection were discarded.

Of those that made it to harvest, some didn't survive winter storage. A very few didn't taste good. One tasted like fresh-mowed grass. (In retrospect, I wish I had kept that one for its prank value alone.) A few that made it through all those steps didn't manage to grow any tubers in the next year.

I did end up with a handful of varieties which seem to grow well enough in my environment, produce tubers that store well, and importantly also taste good. A selection of my varieties can be seen in the photos above. Some even ended up having very nice flowers, though one would never mistake the plants for garden flowers. This year I'm planning to grow a commercial type along side mine, to get a better sense for how well they produce vs the commercial control. Every year in the garden is a new experiment.

References:

• Grafted tomato/potato
• www.territorialseed.com/product/grafted-tomtato-ketchup-n-fries/new_for_spring_2015
• www.youtube.com/watch?v=6V_X77CeTcs
• Solanine
• www.food-info.net/uk/qa/qa-fp95.htm
• www.smithsonianmag.com/arts-culture/horrific-tales-of-potatoes-that-caused-mass-sickness-and-even-death-3162870/?no-ist
• Tom Wagner
• www.tomatoville.com/showthread.php?t=6761
• gardenrant.com/2012/01/an-interview-with-the-true-potato-seed-man.html
• Potato Seeds
• www.tom8toes.com/index.php/potatoes.html
• tatermaterseeds.com/shop/index.php?main_page=index&cPath=2&zenid=c96f378a548b3b7031d204e6f608f617 
• www.doublehelixfarms.com/true-potato-seed-TPS
• www.cultivariable.com/

The Color of Beans 3

A great deal of research has gone into our understanding of how colors are made in plants. I've previously written about the carotenoid pigment pathways in tomatoes [1] and peppers [2], condensing a great deal of published literature in the process. Until recently, I didn't have a solid grasp of the pathway plants use to make a second major category of pigments, the flavonoid pigments. These pigments are responsible for many of the red/purple/blue colors you see in flowers and other plant parts, but I've been learning about them through my focus on the various colors of dry beans.

The carotenoid pigment pathway I discussed in those earlier articles was relatively simple. A single main pathway, with a couple branches. The anthocyanin pathway figure at above-right is a bit more complicated. The figure is a consensus pathway, built from research in a few different species. There are definitely more pieces that could be added, but this amount is a good start. The colored highlights are intended to represent the colors of those chemicals. The lower red, orange, and blue pigments are anthocyanins, the pigments responsible for the color of many flowers (and other plant parts). The white-to-brown gradient highlight is for the proanthocyanidins. They oxidize over time, changing from clear to brown. The red pigment at upper-left is found in some trees, but I wasn't able to find too much information about them. The yellow pigments at right are found in various plants and plant parts, but they're not generally the source for bright yellows in flowers. (The enzyme FGT leading to astragalin at far right is something I made up, since I couldn't find any research naming the enzyme performing that step.)

At left is a heavily reduced version of the first figure, trimmed to an approximation of what seems to be going on in common beans (Phaseolus vulgaris). Combinations of the yellow, red, blue, and brown pigments seem to be responsible for most of the variations in color that we see in dry beans. I've seen some evidence for a brown pigment derived from the yellow ones here, but I haven't found any research clarifying the chemistry involved. There's the possibility of some green pigments made up from a different metabolic pathway, but I haven't found sufficient research about them to know if they're represented in beans.
Various of the trimmed compounds are also found in common beans, but they don't seem to be found in significant amounts. The orange pelargonidin pigments have been reported in some bean varieties, but I've never come across a common bean that has a color dominated by orange pigment. There might be orange examples from P. coccineus, the scarlet runner bean, but I'm still investigating this.

---

The colors of beans drew attention far before we had any understanding of the physiology of the pigments involved. Much of the early published research into bean colors sought to identify different genes responsible for the traits. Eventually the gene labels assigned by different authors got correlated with each other and the set of labels for important color genes became standardized. Even more recently, there have been efforts to identify the molecular mechanism behind the different classical gene labels. Some gene labels are now associated with specific enzymes or other genes important in the flavonoid pathway.

• R [red] : Enzyme F3'H, or more likely a transcription factor driving F3'H in the seed coat. F3'H is important for stress response in plant tissues and so is unlikely to be absent even when the enzyme isn't active in the pathway.
• V [violet] : Enzyme F3'5'H. This one isn't as important as F3'H and is entirely absent in many plants.
• J : Pretty solidly identified as the enzyme DFR.
• P : A transcription factor driving expression of several genes important in the flavonoid pathway. In the figures above, the regulated enzyme targets are drawn in blue.
• B : A transcription factor driving expression of chalcone synthase (CHS) and/or chalcone isomerase (CHI).
• G : A transcription factor leading to increased levels of astragalin, perhaps by driving expression of FLS and/or FGT. Likely has other impacts, but I haven't found sufficient research.

Tracking down which gene was associated with which step in the pathway was tricky. Many of the older papers had models for what a given gene did, but then those models were overturned by more recent research. The paper identifying V as being the gene for the enzyme F3'5'H was only published in March 2022. Finding that paper got me interesting in trying to see how many of the others could also be associated with a specific part of the pathway. The other gene notes above came from the scattered papers linked in the references section, though few were specifically the point of the papers.

My goal was to better understand what the gene labels were doing, so I could better figure out what genes were likely to be involved in the beans I was growing and crossing. I'll write more on that another time.

References

1. Related blog posts:
1. https://the-biologist-is-in.blogspot.com/2014/04/the-color-of-tomatoes.html
• Carotenoid pigments in tomatoes.
2. https://the-biologist-is-in.blogspot.com/2015/11/the-color-of-peppers-2.html
• Carotenoid pigments in peppers.
3. https://the-biologist-is-in.blogspot.com/2018/10/the-color-of-beans-1.html
• Introduction of my #BlueBeanProject.
4. https://the-biologist-is-in.blogspot.com/2022/12/the-color-of-beans-2.html
• Status update of my #BlueBeanProject.
5. https://the-biologist-is-in.blogspot.com/2019/11/biology-of-blue.html
• Discussions around the chemistry of blue in biology.
2. Papers related to anthocyanin pathway in bean, cotton, etc:
1. http://arsftfbean.uprm.edu/bic/wp-content/uploads/2018/04/ChemistrySeedCoatColor.pdf
2. https://nph.onlinelibrary.wiley.com/doi/full/10.1002/ppp3.10132
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3602603/
4. https://link.springer.com/article/10.1007/s11738-011-0858-x
5. https://pubmed.ncbi.nlm.nih.gov/28981784/
6. https://bmcplantbiol.biomedcentral.com/articles/10.1186/s12870-019-2065-7
7. https://pubmed.ncbi.nlm.nih.gov/35289870/
8. https://squashpractice.com/2011/10/08/bean-genes/
9. https://journals.ashs.org/jashs/view/journals/jashs/124/5/article-p514.xml
10. https://www.frontiersin.org/articles/10.3389/fpls.2022.869582/full#ref33
11. https://journals.ashs.org/downloadpdf/journals/jashs/120/6/article-p896.pdf
12. https://journals.ashs.org/downloadpdf/journals/jashs/125/1/article-p52.pdf
13. https://www.semanticscholar.org/paper/Allelism-Found-between-Two-Common-Bean-Genes%2C-Hilum-Bassett-Shearon/f9cef3175289b7d2822461b9d495d8885bb67a48
14. https://www.semanticscholar.org/paper/Inheritance-of-Reverse-Margo-Seedcoat-Pattern-and-J-Bassett-Lee/7557538290b700d1fd980a24fba3148846861690
15. https://www.semanticscholar.org/paper/The-Margo-%28mar%29-Seedcoat-Color-Gene-Is-a-Synonym-%28-Bassett/d1c58ec1fa0bf9e500d8bd48364a61568b0b7a11
16. https://naldc.nal.usda.gov/catalog/IND92036951

The Color of Pineapple

The pineapple we all grew up with is a bright yellow color. The pineapples of today isn't necessarily the same shade. Del Monte is now selling a variety with a distinctly pink flesh, called PinkGlow^TM or Rosé pineapple. This is a bio-engineered variety, first conceptualized way back in 2005. A patent for the variety was issued in 2012 and the US FDA deregulated the variety in 2016, deciding the variety was essentially the same as other varieties with regards to safety and regulatory concerns.
The variety started as an extra sweet variety grown in Hawaii called MD2. This pink version shares the extra sweet and low acid traits of that original variety. I think it is a worthwhile product, even though (in my limited experience) most people's reactions to seeing the pink cut pieces at left was to think they looked like pieces of meat.

---

The patent is a bit of a pain to read, as they generally are. The "DETAILED DESCRIPTION OF THE INVENTION" section is where they describe the details of the alterations they made.

At left is a sketch of the carotenoid pathway in pineapples. There is limited published information about the specifics of the pathway in pineapple, so this diagram was constructed from more general research in tomatoes, peppers, and other species. At right is a closeup of the pathway altered to illustrate the changes that were made in the pink pineapple, as described in the patent.

The first modification was to introduce a second copy of the phytoene synthase gene, driving increased amounts of metabolic energy through the carotenoid pathway. This is represented in the figure by a larger arrow at the top. The added gene was combined with a pineapple fruit flesh specific promotor, so the rest of the plant doesn't have its carotenoid pathway messed around with.

The second modification was to shut down two enzymes, lycopene beta-cyclase and lycopene epsilon-cyclase, normally responsible for converting lycopene into the next steps in the two branches of the carotenoid pathway after lycopene. The consequence of this is all the metabolic energy passing through the pathway is stopped at lycopene. Shutting down these genes was performed by RNA interference (RNAi), also driven by a copy of the same fruit flesh specific promotor. Again, this prevents the modification from interfering with the carotenoid pathway elsewhere in the pineapple plant.

The carotenoid pathway is important for a plant's stress response and other systems. It is likely a pineapple plant would survive more dramatic alterations to the carotenoid pathway that impacted the entire plant, but doing so would throw off the existing balance. The efforts they've taken to limit the pathway tweaks to only happen within the fruit flesh were important to ensure the plants generally are as productive and healthy as the pineapple they started with.

---

A third modification was atempted, but how the patent is written indicates they're not exactly sure the alteration worked. Commercial pineapple production relies on precision planning. To get a pineapple crop to mature at a specific planned time, the plants are treated with a hormone which induces flowering. In pineapple, the hormone that triggers flowering is the simple gas ethylene. Either ethylene or the similar shaped molecule acetylene is used to induce a crop to start blooming at a specific time. The problem is, pineapple plants will initiate blooming all on their own, when the growers may not want the plants to do so. This is called "natural flowering" and interferes with the plans of the growers.

So, to try and reduce the rate of natural flowering, the third modification was to try and supress the ACC synthase gene important for normal ethylene biosynthesis. They again used RNAi for this, targeted to growing meristems where the gene enzyme activity is important for normal flower induction. I suspect the reason the patent expresses uncertainty about this modification working is because at the time of patent filing, they didn't have enough experience with growing the new pineapple in field conditions to be able to see a reduction in the rate of natural flowering. By now they'll know for sure if it worked.

References:

1. Marketing piece: https://specialtyproduce.com/produce/Pinkglow_Pineapple_17105.php
2. Patent: https://patents.google.com/patent/USPP25763
3. FDA statement: https://www.fda.gov/food/cfsan-constituent-updates/fda-concludes-consultation-pink-flesh-pineapple
4. Carotenoids in tomatoes: https://the-biologist-is-in.blogspot.com/2014/04/the-color-of-tomatoes.html
5. Carotenoids in peppers: https://the-biologist-is-in.blogspot.com/2015/11/the-color-of-peppers-2.html
6. Pineapple flower induction: https://www.echocommunity.org/resources/f0e9cfeb-ba1d-435e-a515-7705ca79b409