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.

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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.

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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.

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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