Better Chemistry Through Breeding

I recently had the opportunity to visit the fabled heart of the USDA-ARS empire: Beltsville.

I heard all about the tornado that knocked down all the campus trees, smashed in the greenhouses and threw doors down hallways a few years ago, visited their food sensory lab (a controlled environment where fruit samples are passed through a wall to waiting taste testers), and saw greenhouses packed full of cacao (where research on one of my favorite fungi, Crinipellis perniciosa, is co-funded by M&M Mars Inc.).

But I was there mostly to visit the pepper breeding program.

One of the goals of this particular program is to breed baby bell pepper (C. annuum) varieties that are equally ornamental and edible. It was an amazingly vibrant greenhouse scene in the middle of a brown winter day: bright green, dark purple and variously variegated bushes of leaves tufted from twisted woody stems and were spotted with flowers and fruit of most colors imaginable. It was a striking demonstration of segregating traits and the heart of what plant breeding is all about. Appropriately, these guys have released a few varieties to the public (including the striking Black Pearl).

The Colors of Peppers

Plants are green due to chlorophylls, orange and red thanks to carotenoids and red, purple and blue (sometimes yellow or orange) because of anthocyanins.* Anthocyanin pigments (actually dyes) are the reason why stressed plants tend to turn purple - especially when exposed to too much sun. Like other plant pigments, they also play important roles attracting pollinators and seed dispersers and protecting the plant from stresses (in this case, UV light) by acting as antioxidants.

The typical bell pepper (C. annuum) is a green plant with green fruit that gradually turn orange, then red as they ripen. In the process, chlorophyll is broken down in the fruit as carotenoids accumulate. Although you won't likely see these varieties in stores, C. annuum can also produce immature fruit that are dark purple or almost black (containing normal chlorophyll and anthocyanins) or violet (lacking chlorophyll but containing anthocyanins). Either way, the black or purple fruit will turn orange and red as carotenoids replace chlorophylls and (in this case) anthocyanins. There are also varieties that lack chlorophyll (and anthocyanins) in immature fruit - and therefore start from white or yellow-green and ripen through orange to red. It's a pretty powerful visual statement to see rows of pepper plants with masses of mixed green-orange-red, white-orange-red or purple-orange-red fruit. The dark purple and violet-fruited peppers also tend to have dark purple foliage and if they happen to contain the genetic locus for foliar variegation, the whole plant may be a mix of purple and white (see first picture). Oh, and the flowers can be purple too (instead of the normal cream color).

So in summary:

• Leaves can be green, violet, black or variegated cream
• Fruit ripen from green, cream, yellow-green, violet or purple through orange to red
• Flowers may be cream or purple!

A Pigment Called Delphinidin

In addition to being surprisingly ornamental, chili peppers turn out to be a great model for anthocyanin development in plants. Structurally, anthocyanins consist of a 3-ring core (the "aglycone" anthocyanidin) that's ornamented with any number of glycosyl (sugar), acyl, methyl and hydroxyl functional groups. While the aglycone component is fundamentally responsible for color, it's also positively charged and inherently unstable at neutral pH. Functional groups not only help stabilize these anthocyanins, but also play important roles modifying the hue and intensity of color (along with associated co-pigments and the pH and metal ion concentration of the relevant cellular compartment). While this produces an extremely diverse and dynamic chemical family, there are only three primary aglycone anthocyanidins: cyanidin, pelargonidin and delphinidin.

Delphinidin anthocyanins are usually the most blue of these due to their large number of hydroxl groups. The absence of delphinidin anthocyanins in most roses, carnations, chrysanthemums and lilies is the reason why blue versions of these flowers have long been lacking. These molecules also become bluer as they receive aromatic acyl groups, are stacked with flavone and flavonol co-pigments and metals and are subject to neutral/alkaline pH. Therefore, even though tulips have no shortage of delphinidin anthocyanins, the intracellular environment prevents these molecules from appearing blue (in this case, due to iron levels). Similarly, the well-known ability of Hydrangea macrophylla flowers to change from pink to blue with soil pH is due to the pH-mobilization of delphinidin-influencing aluminum ions.

The chain of enzymes that are needed to build anthocyanins have been known for some time, but as is often the case, we're still figuring out exactly how different regulatory mechanisms manage to fine-tune the flow of precursors through the various steps of the biosynthetic pathway. In this case, the purple color is almost entirely due to a single anthocyanin:

delphinidin-3-p-coumaroyl-rutinoside-5-glucoside

(It's probably a safe bet where the aglycone was first discovered)

While a whole series of enzymes are needed to build this delphinidin glucoside, whether the purple end product is actually present in a given C. annuum variety is determined by the A locus, which encodes a MybA transcription factor gene.** This MYB protein is thought to bind with a particular WD40 protein, which in turn binds to a MYC protein to form a single protein complex that works together to turn on one of the specific enzymes in the anthocyanin biosynthetic pathway. A naturally occurring mutation in MybA apparently changes the property of this protein such that this regulatory MYB-WD40-MYC complex fails to assemble (and no purple is made!). Quantitative variation in the amount of purple in peppers that contain a functional version of MybA is controlled by an additional gene, moA (aka modifier of A). However, neither the identity of moA nor the more complex regulatory structure that controls purple accumulation in leaves seems to have been figured out yet.

So what's next for chili peppers? Lightbourn et al. suggest that as there are no pH differences between purple and black-fruited varieties, there're might be an opportunity to create blue peppers by selecting parents with more alkaline pH (to modify the hue of the delphinidin anthocyanin from purple to blue). There are already 2 known genes in petunia that modify vacuole pH. As both petunia and peppers are in the same family (Solanaceae), there's a chance it'd be pretty straightforward to find a pepper version of these genes to make the intended changes. 

There have been lots of attempts to engineer anthocyanin synthesis, largely in flowers and mostly unsuccessful. Early on, it was expected that some simple changes would allow major color changes to be made to plants (e.g. "blue" roses), but for the most part it's turned out to be trickier than hoped. In hindsight, it shouldn't be so surprising considering the extent to which color is influenced by pH, metal ions and various pigments and co-pigments (that often have to be shuttled among different cellular compartments). It's difficult enough with current technology to optimize the expression of multiple genes at one time, but the complications of biochemistry really push the envelope. 

Even if some very talented scientist-artist manages to engineer a flower with really unique coloring, it's unlikely we'd see it in our gardens anytime soon. The path to commercialization is just soaked with regulation, all of which differs from country to country (which is even more complicated due to the frequency with which flowers are often grown, warehoused and sold in different countries). Existing regulations are already pretty burdensome for major traits in major crops where your chance of recouping your expenses are at their best. The floriculture market is incredibly segmented and the sales per any one cultivar are relatively small. Until regulation gets streamlined, no company is likely to be bankrolling any sci-fi new flowers.

    Lightbourn, G., Griesbach, R., Novotny, J., Clevidence, B., Rao, D., & Stommel, J. (2008). Effects of Anthocyanin and Carotenoid Combinations on Foliage and Immature Fruit Color of Capsicum annuum L. Journal of Heredity, 99 (2), 105-111 DOI: 10.1093/jhered/esm108
    Stommel, J.R., & Griesbch, R.J. (2008). Inheritance of Fruit, Foliar, and Plant Habit
Attributes in Capsicum J. Amer. Soc. Hort. Sci., 113 (3), 396-407
    Stommel, J.R., Lightbourn, G.J., Winkel, B.S., & Griesbach, R.J. (2009). Transcription Factor Families Regulate the Anthocyanin Biosynthetic Pathway in Capsicum annuum. J. Amer. Soc. Hort. Sci., 134 (2), 244-251
    Tanaka Y, Brugliera F, Kalc G, Senior M, Dyson B, Nakamura N, Katsumoto Y, & Chandler S (2010). Flower color modification by engineering of the flavonoid biosynthetic pathway: practical perspectives. Bioscience, biotechnology, and biochemistry, 74 (9), 1760-9 PMID: 20834175

* Plants in the Caryophyllales (beets, swiss chard, cacti and many carnivorous plants) rely on betalains (which are red or yellow) instead of anthocyanins - and no plant is known to be capable of making both. 
** chalcone synthase -> chalcone isomerase-> flavanone 3-hydroxylase-> dihydroflavonol 4-reductase-> anthocyanidin synthase -> UDP-glucose-flavonoid-3-O-glucosyltransferase
*** As usual, my reports of what other scientists are up to is limited to what is publicly available. Don't want to cause someone to get scooped!