Colors Within: A Study of the Pigmentations in Deciduous and Broadleaf Evergreens

Part of the Young Naturalist Awards Curriculum Collection.

by Megan, Grade 9, Texas - 2008 YNA Winner

"Autumn is a second spring where every leaf is a flower." —Albert Camus 

I've always been fascinated by the seemingly magical transformation that leaves go through during the autumn. Between the magic and my curiosity, it seemed natural to investigate this change of color.

Diagram showing plastids with colored pigments.

My investigation led me to some of the hows and whys of the changing colors of leaves. I learned that leaves have pigment-containing plastids in the specialized organelles found in plant cells. These plastids make it possible for photosynthesis to occur and provide the plant with food. The plastids contain different pigments that give the leaf its color. The pigment located in the chloroplasts is the one we see in most leaves most of the time. This pigment is chlorophyll, which is green. But leaves contain other pigments, and they are located in the chromoplasts. These pigments include xanthophylls (yellow), carotenoids (yellow, orange, red), and anthocyanins (red). These colors are normally hidden by the chlorophyll except for when the chlorophyll begins to break down during the autumn season. This is when we can then see the yellow, orange, and red pigments.
Having lived in South Dakota, where the trees burned with color, I noticed some trees and bushes in Texas never changed color nor lost their leaves during the fall. I learned from our Tom Green County extension agent, John Begnaud, that seed-producing plants are called angiosperms, and that the trees and bushes I was interested in were either deciduous angiosperms (which lose leaves in winter) or broadleaf evergreen angiosperms (which keep leaves all year). The broadleaf evergreens, then, are not to be confused with evergreen trees, which are coniferous or cone-bearing trees, or what we often refer to as pine trees.

Determining the GPS of sample B.

During the growing season of deciduous trees, when the chlorophyll is the only pigment visible, the other pigments—the xanthophylls, carotenoids, and anthocyanins—are all present. When the plant goes into its dormant stage, the chlorophyll starts to break down, and thus we see the beautiful colors that don the trees during the fall.
Problem and Hypothesis 
Because we usually see the xanthophylls, carotenoids, and anthocyanins only when deciduous plants are getting ready to lose their leaves, I wanted to know if these pigments were also present in broadleaf evergreens. I wanted to test the plants to see what pigments they had in their leaves compared to the deciduous trees.
I could see the yellow, orange, and red pigments in the deciduous trees as the chlorophyll broke down in the fall, so I knew they would show these pigments when I did my testing. But would these pigments show up in broadleaf evergreens that do not change color or lose their leaves in the fall? If the pigments are present, there is no visible evidence.

Deciduous Samples.

In my search into the reason for these pigmentations, I learned that carotenoids and xanthophylls work as accessory pigments that help chlorophyll absorb light. More specifically, the pigments capture wavelengths in the light spectrum that chlorophyll cannot absorb.
Because the accessory pigments seemed to be important in the well-being of a plant, I believed that the leaves from both the deciduous and the broadleaf evergreens would contain all the pigments: chlorophyll, xanthophylls, carotenoids, and anthocyanins. Because all the leaf samples were visibly green, I believed they would definitely contain the pigment chlorophyll, which I believed would be the most prominent of the pigments.

Crushing leaf samples.

I also believed that the pigments in the deciduous plants would be more intense than the pigments in the broadleaf evergreens. I thought this because the pigments in the deciduous plants appear as vibrant colors during autumn season, but the broad leaves of the evergreens remain green.

Materials and Methods 

Because I was testing to find out if broadleaf evergreens and deciduous plants contain all four of the pigments, I had to collect a good representation of both broadleaf evergreen and deciduous plants as specimens. So on a warm sunny day in September, I set off to collect leaves throughout my hometown of San Angelo.

Coffee filter strips soaking in solution.

On my first outing, I collected what I believed to be six deciduous leaf samples. I identified the specific location of each sample using a GPS (global positioning system) and labeled the samples 1 through 6.

Promptly after collecting the samples, I crushed the leaves by twisting and tearing them to release the pigments in the plant cells. I put each sample's crushed leaves into individual 16-ounce glass jars and poured enough 91% isopropyl alcohol into each jar to cover the crushed leaves well. I boiled tap water and poured it into a 9-by-13-inch baking pan and placed the jars in the hot water to help with the extraction of the pigments from the leaf cells. I let the jars sit in the pan of hot water overnight.

Six different branch specimens, each labeled "A", "B", "C", "8", "E", and "F".
Broadleaf Evergreen Samples.

I had decided the best way to test my hypothesis was to use a paper chromatography test. Paper chromatography works by first using a solvent, in my case isopropyl alcohol, to create a solution, which I did when I crushed my leaf samples and added alcohol. The solution then moves up the strip of chromatography paper by capillary action to separate the molecules of pigments according to their molecular structure. I started off using strips of coffee filters, as many chromatography paper methods suggested. I taped a strip inside the side of each jar so that just the edge of the paper was submerged in the solution, which allowed the solution plenty of room to move up the chromatography paper and for the pigments to separate. I covered the jars and let them sit overnight. I then removed the coffee filters and let them dry.
But I was not happy with the results. Although the coffee filters test showed some separation of pigments, they weren't as intense as I desired. I decided to repeat the chromatography test using drawing paper, hoping that it would have a better showing of the intensity and separation of the pigments. But again, I was not satisfied.

Deciduous Sample Results.

I needed something that promoted a stronger capillary action, something more absorbent. I then used Viva paper towels as my testing strips, and I was very satisfied with the results. They showed the separation of pigments and more intensity in their colors. I let the strips of papers dry and recorded my results.

To make sure I was getting broadleaf evergreens, I waited until January to collect my evergreen samples. I collected six evergreen samples and used a GPS to record the locations of each plant and a light box to photograph the samples taken. I also identified these samples, with a little help from my friends. I used A-F to label my list of my broadleaf evergreen samples: A) Texas mountain laurel, B) Texas sage, C) yucca, D) live oak, E) palm, and F) viburnum.

Broadleaf Evergreen Sample Results.

I repeated the same tests I had done with the deciduous samples. I used all three types of chromatography paper to keep my results accurate. I let the strips of papers dry and recorded my results. I began to compare and analyze my samples.

Results and Conclusions 

Overall, the results in comparing the deciduous plants and the broadleaf evergreens were very similar. Both the broadleaf evergreens and the deciduous plants contained the pigment chlorophyll, as well as xanthophylls, carotenoids, and anthocyanins. All the samples in the deciduous group showed all pigments. All the samples in the broadleaf evergreen group, except the yucca, contained all the pigments as well. But what I found fascinating was that the broadleaf evergreens showed a higher intensity of pigments than the deciduous. This was the opposite of what I hypothesized.

Mexican White Oak showing signs of stress.

I did find some interesting details within my overall results. Sample 1 (Mexican white oak) was from a deciduous plant and was significantly lower in the intensity of its bands of pigment than the rest of the deciduous tests. I observed that the specimen was quite distressed, and that could have been the reason for the lack of pigment intensity.

Because of the significantly lower pigment intensity in the sample of the Mexican white oak that I believe was stressed in some way, I wondered if it would be possible to apply this technique of checking pigments in leaf samples as a way of checking the health of plants. Because the pigments work as accessory pigments that help absorb light, they are very important to the well-being of the plant. If a plant is lacking these accessory pigments, it could be an internal sign that the plant has become stressed for some reason. A chromatography test could possibly be used to determine if a plant is lacking any pigments before there are any visible outward signs of stress.

Pigments show less intense than all other results.

The yucca, which is a broadleaf evergreen, seemed to show no signs of any other pigments except for chlorophyll and maybe a bit of yellow pigment.

My results showed that chlorophyll, xanthophylls, carotenoids, and anthocyanins were present in both the deciduous and the evergreen plants, with the pigments in the broadleaf evergreens being more intense than the pigments in the deciduous plants. From research and observation, I concluded that this could be because I tested the deciduous samples during the autumn season. During that time of year, the pigments in a tree are breaking down, while during a tree's growing season the chlorophyll is constantly being replaced because of the need for photosynthesis to make food.

Yucca results showing mainly chlorophyll.

I would like to test the same samples of deciduous trees during their growing season to see if there is an increase in the pigment intensity. I would also like to test the broadleaf evergreen trees throughout the year. Would their pigments be more intense in the spring when we see new growth? Or would the intensity of the pigments remain the same? How would drought affect the pigments? What would they be right after a freeze? Is there a way to measure the exact amount of the pigments in the leaves? And is there a way to test for pigments without having to destroy them? All of these questions lead me to dig deeper into the magic of pigments and broaden my study.

So my hypothesis was proven partially correct. I stated that I believed that both the broadleaf evergreens and the deciduous leaves would contain all the pigments, and they did. I also stated that chlorophyll would be the most prominent pigment because all the leaves were visibly green. This also appeared to be true.

The last leaf of autumn.

But my thoughts about the intensity of the pigments between the deciduous plants and the broadleaf evergreens proved incorrect. It was the broadleaf evergreens that showed a higher intensity of pigment color, even though these pigments were not outwardly visible in the broadleaf evergreens as they are in some deciduous plants. It seems that all the leaf samples contained the colors we enjoy each autumn.

As the last of the autumn leaves falls to the ground, and the vibrant colors of the season begin to fade away, the trees are preparing for a whole new season of life to begin. The change of colors during this magical season is more than a beautiful landscape, for it is the colors within that provide the food for life itself.


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