Hot Peppers under the Microscope

Something new under the sun… pepper pigment packaging.*

pepper

Just when you thought you had seen the last of red hot chile peppers in the Superbowl halftime show yesterday**, I’m still talking about Capsicum science. The other pepper-themed posts have been all about the heat, but capsaicin isn’t the only chemical these plants make. Capsicum species are also great at producing nutritious carotenoids. Again, because Capsicum species grow well in arid environments, they offer an advantageous platform for providing these nutrients in an efficient way. In order to get our peppers to pack more punch in the nutrition department, we must better understand the metabolic machinery that makes these colorful molecules.

Carotenoids are the pigments that give us the yellow, orange and red colors in our peppers. You may be familiar with the common beta carotene molecule that is the precursor for vitamin A. That’s just one pigment. Plants and peppers, in particular, are adept at making a wide range of colorful carotenoids. However, all peppers start out green because the cells of the fruit contain photosynthetically active chloroplasts. As the fruit matures, these green chloroplasts undergo a major developmental change to become colorful chromoplasts. This process involves changes in gene expression, protein function, membrane structure and overall metabolism. By the end of the transition, chromoplasts are filled with an array of carotenoids, giving the fruit its hallmark red color.

Today’s journal club features a recent paper by Kilcrease et al that explores pigment localization within the chromoplasts of living plant tissue. This combines the Capsicum expertise of New Mexico State and the hyperspectral imaging capabilities of the Timlin lab at Sandia National Labs.*** It turns out that the different pepper varieties make characteristic carotenoids and these are made/stored in specific intracellular sites.

Here’s how the science breaks down:

Observations: For this example, observations are coming from two different directions (pepper pigment biology and spectral imaging method development).

Different pepper varieties produce different arrays of carotenoid pigments in their mature fruit. The literature suggested that there were significant differences in chromoplast morphology among these varieties. Some experimental evidence suggested that certain pigments were so concentrated they formed crystals within the plant cells.

Hyperspectral confocal Raman microscopy**** can provide chemical information in high resolution on living plant tissue. The molecular structure of chemicals like carotenoids makes them straightforward to identify. Using multivariate image analysis, a spatial model can be generated to show where the chemicals are within the microscopic image.

Hypothesis: Hyperspectral confocal Raman microscopy can be used to determine the carotenoid localization within the ripe fruit of different pepper varieties. This data will show whether or not the pigments are localized in distinct places within chromoplasts.

Experiment: Researchers analyzed the tissue of 5 different hot pepper varieties (mature fruit; and no, they didn’t use Bhut Jolokias or Scorpion peppers) using four different microscopic techniques (scanning electron microscopy, transmission electron microscopy, laser scanning microscopy, and hyperspectral confocal Raman spectroscopy) to identify subcellular localization of carotenoid pigments. The pigment content of each of the peppers was also analyzed using the analytical chemistry technique HPLC.

Results: The different types of peppers analyzed in this study varied considerably in carotenoid composition, chromoplast structure and sublocalization of carotenoids. The pigments did localize to specific sites within the chromoplast as well as some subcellular lipid bodies outside of the chromoplast.

Conclusions: The combination of these methods allowed for the more complete characterization of chromoplast structure and pigment composition in five different pepper varieties. These data can serve as new traits when considering breeding peppers for increased nutrient content.

Think Ahead:  The example the authors give is for aiding in the breeding of superior chiles in terms of increased carotenoid content. For example, a variety with high carotenoid content can be crossed with one with large chromoplasts to potentially yield offspring with even more carotenoids filling the larger chromoplasts. In this way, the results from these analyses will provide new molecular traits characteristic of certain pepper varieties. Genetics can then be used to mix and match those traits in desirable ways. Also, all of these experiments were performed on fruit that was already at a certain stage of ripeness. It will be interesting to perform an extended analysis on fruit as it ripens from green to red. This kind of time course experiment will yield more information about how these specialized synthesis and storage sites for the different carotenoids form as the chromoplast develops.

Ultimately, knowing more about how peppers make their carotenoids will allow scientists and breeders to develop more nutritious plants. This means not only understanding the chemical synthesis of these molecules, but also how the plant cells physically/spatially accommodate the increase in those metabolic pathways.

Johnna

*Say that fast 3x!

**It was a weird pairing of performers, but ‘Give it away’ still rocks IMHO.

***The second author on this paper is Aaron Collins (Sandia National Labs), an old grad school and photosynthesizer colleague of mine from WashU. He mentioned this project to me at a meeting last summer and bragged about the benefits of collaborating with biologists working on Capsicum (=pepper perks!).

****Yes, as fancy as it sounds.

References and Links:

(Caution: paywall for full text) http://www.ncbi.nlm.nih.gov/pubmed/24118159

Here’s a nice research highlight with the Raman microscopy figure) http://parc.wustl.edu/files/parc/Timlin_PARC_EFRC_Research_Highlight_September_2013.pdf

For more on hyperspectral imaging: http://www.sandia.gov/bioenergy-biodefense/timlin.html

For more on chile peppers: http://www.chilepepperinstitute.org/

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