Written by Aida Martinez
All organisms require a metabolic process that allows them to convert something they intake into energy. To convert light into energy, all organisms contain tetrapyrroles (involved in energy transfer, catalysis and transduction) (Reinbothe 1995). Of the greatest sources of energy is photosynthesis. Photosynthesis is a metabolic process (present in photosynthetic organisms) that intakes light, carbon dioxide, minerals, and water to produce chemical energy such as sugars, ATP and oxygen. Sugars and ATP are stored in the organic molecules of the organism to be broken down later. In other words, the carbon derived from the carbon dioxide or present in water is kept by the organism to produce sugars and other macromolecules. Without the help of pigments, photosynthesis cannot continue its process. Pigments are molecules that help absorb energy in the form of light. All photosynthetic organisms contain different pigment types that absorb different wavelengths of light and reflect a different color on their surface. There are three main types of pigments: chlorophylls (a and b), carotenoids, and phycobilins. Carotenoids and phycobilins are considered “accessory pigments” which capture energy in the form of photons and then transfer it to chlorophylls. However, all eukaryotic organisms have chlorophylls because it is the only pigment that converts light into chemical energy.
Whether an organism is exposed to light or not can have a significant impact in pigment composition. Indeed, a study confirms that cultivations carried under poor light (artificial light in this case) present a higher biomass of chlorophyll present in the organism (Nicula et al 2012). Chlorophyll absorbs blue and red light in turn giving a plant its green reflective color. In this experiment, light grown barely and dark grown barely visually have different colors on their leaves (light grown barely looks green) while dark grown barely is more yellow/brown. The objective of this first part of the experiment is to determine how the environmental conditions of light affect the color pigmentation of the plant and so give it its unique color. The null hypothesis for this experiment is: the presence of light exposure does not influence pigment composition of light grown barely and dark grown barely.
Evolution also has significance in pigment composition variation. If an organism has the same or almost the same amount of pigments, there may be a common ancestry between both organisms. Indeed, chlorophyll, the most abundant and present pigment in photosynthetic organisms, is said to be found only in two of the three main domains of life: bacteria and eukaryotes (Blankenship 2010). The idea of how the evolutionary history of an organism affects its photosynthetic pigment composition can be tested by comparing different organisms. In this experiment, barely (a eukaryote) will be compared to more primitive form of eukaryotes: algae (red, brown, and green) and cyanobacteria to see if they have the same composition or very similar pigmentation. The null hypothesis is: brown, red, green algae or cyanobacteria pigment each show pigmentation differences compared to barely pigments and are therefore all evolutionary unrelated to barely.
Materials and Methods
In order to test both null hypotheses, two separate experiments must be made to satisfy both objectives. Experiment 1A-1B is done to obtain data that will help us understand if there are any significant environmental conditions that give rise to the difference between the pigmentation of dark grown barley and light grown barely. This experiment (part 1A-1B) will obtain spectrophotometer data that will determine the absorption rates at a wavelength of the visible eye wave spectrum for both dark grown barely and light grown barely. The absorption spectra of each organism can be compared to see if there are any common peaks at a particular wavelength signaling different or same pigmentation. Experiment 2A-2B, is done to satisfy a different objective: to determine which photo synthesizer of the four different used (red algae, green algae, brown algae, and cyanobacteria) are evolutionary most related to barely. A spectrophotometer analysis is made to each of these organisms to determine absorption peaks, if any, similar to the absorption peaks of barely (done in the first experiment 1A-1B). Both experiments will use TLC (Thin Layer Chromatography) to separate the organic compounds of each organism and determine the different pigments present in each.
This part of the experiment focuses on dark grown barely genus and light grown barely both genus Hordeum. First, a sample of 4 leaves of light grown barely and 3 ml of methanol were placed on a glass mortar with a pestle. The leaves were crushed for 3 minutes until the liquid became dark green and most of the fibers were evenly mixed in the liquid. A clean microcentrifuge tube was obtained and was pipette 1 ml (1000 micro liters) of the barely crushed liquid. The mortar must then be cleaned and the same procedure was repeated with dark grown barely leaves. The crushed liquid was placed in a different new micro centrifuge tube. Both microcentrifuge tubes were placed in the centrifuge for 5 minutes. After, two new cuvetes were obtained and one was pipetted 500 micro liters of light grown barely buffer(cuvete 1) and the other pipette with 500 micro liters of dark grown barely phosphate buffer(cuvete 2). 50 micro liters of centrifuged light grown barley were then pipetted to the corresponding cuvete 1 and 100 micro liters of dark grown barely were added to the other corresponding cuvete 2. Cuvetes were covered with parafilm, mixed by tilting, and taken to the spectrophotometer (coordinated by the undergraduate student lab assistant). A printout of the absorption spectrum was given.
A TLC experiment was done for this second half of the first experiment. Obtained were two capillary tubes. One tube was placed on the methanol extract of the light grown barely. The other was placed on the methanol extract of the dark grown barely. A TLC strip was obtained and mark 0.05 cm from the bottom up and top down with a pencil (on the silica side). Using the capillary tube for that liquid, a pencil was used to spot one light grown barely liquid dot on the .05 cm mark. The process was repeated for the dark grown barely. Both were then dried with an air hose. Spotting was repeated for an additional 10 times. Once done, the strip was taken to the fume hood and placed in a “TLC Tank” container with some ml of ethyl acetate: petroleum ether. Once solvent was absorbed to the top .05 cm mark line the strip was taken out immediately and marked any pigment spots using a pencil. Using a cm ruler, the distance was measured from the bottom .05 to each pigment mark. Then the total distance traveled by the solvent was measured. This is used to determine the retention factor of each pigment: distance spot moved/distance solvent moved.
This second part of the experiment now focused on four different organisms: Cyanobacteria (genus Spirulina), Brown Algae (genus Laminaria), Green Algae (genus Ulva), and Red Algae (genus Chondrus). Four different cuvettes where obtained (one for each organism). Each cuvete was pipetted 500 micro liters with their corresponding phosphate buffer. After, methanol extracts of each organism were obtained. Each cuvete was pipetted with 50 micro liters methanol extract corresponding to that organism’s buffer phosphate extract. Each cuvete was then sealed with parafilm and mixed by tilting. Cuvete were then taken to the spectrophotometer. An absorption spectrum graph was printed for each of the organisms. Experiment 2B
The procedure for this part of the experiment is very similar to that of part 1B. A capillary tube was assigned to each of the microcentrifuge methanol extracts. Two strips of the TLC were marked with 0.05 cm from the bottom up and from the top down with a pencil. Spots of red algae and cyanobacteria were placed on the same strip. Green and brown algae spots went on the second strip. There is no particular order of pairing, but for the sake of this experiment, these were paired as indicated. A spot of each organism’s liquid extract was placed on the .05 cm marking. Then it was air dried. The process was repeated for 10 times each strip. Then, strips were individually placed on separate “TLC tanks”. Once the solvent reached the top .05 cm marking, the strips were taken out using forceps and measured at their pigment marks to determine their retention factor coefficient.
Part 1A- 1B
Each major visible peak was identified at the spectrophotometer graph and matched with a corresponding pigment (shown here in parenthesis) using figure 7. The wavelength of each visible peak is also matched with the color spot seen on the TLC.The light grown barely graph shows four peaks at respective wavelengths Figure 1 shows one peak (the highest peak) at wavelength 430 nanometers (nm) with absorbance of .6769 (chlorophyll a), then one peak at 490 nanometers with absorbance .4313 ( Carotenoid Fucoxanthin or B-Carotene), another at 460 nm absorbance .6154(chlorophyll b), and a last peak at wavelength 660 nanometers with absorption .2288 (chlorophyll a). The dark grown barely graph shown on figure 2 has absorption peaks at wavelength 490 at absorption .1390(Carotenoid Xanthophylls). And another peak between 430 nm at absorption .1883 (chlorophyll a). The retention factors of the light grown barely are .9795 cm (a green spot on the TLC), .85 cm (a lighter green spot), .70 cm (a yellow spot), .5875 cm (a faded yellow spot). For the dark grown barely, the retention factors are .90 cm (a visible not too dark yellow spot) and .68 cm (a faded almost absent yellow spot). Other very light hard to see yellow spots may have been shown, however, were not recorded because they were not visible to the naked eye.
There are four total spectrophotometer graphs, one for each organism used: red algae,
brown algae, green algae, and cyanobacteria. Red algae shows four total peaks (figure 3), two of them with high amplitudes at 430 nm with absorption .1803 (chlorophyll a) and the other at 680 (chlorophyll a) nm with absorbance .0510. The other two are not too sharp peaks: one at 490 nm absorbance .1128 (either 3 Carotenoids), and the other at 580 nm absorbance .0477 (chlorophyll c).The brown algae has two visible peaks (figure 4) one at 520 nm with absorption .2296 (Carotenoid Fucoxanthin) and another at about 680nm absorption .1167 (chlorophyll a). Green algae shows two peaks (figure 5) one at 445 nm with absorption at about .5318 (can be a chlorophyll either a or b). The other is at about 660 nm with absorption .1854(chlorophyll a). Lastly, the cyanobacteria shows three peaks (figure 6) one at 430 nm with absorption .5898 (chlorophyll a), another at 580 nm with absorption .1513 (chlorophyll c), and a last peak at 660 nm with absorption .3216 (chlorophyll a). The retention value for red algae is .7831 cm (dark green spot) no other yellow spots were seen with the naked eye. Retention values for brown algae are .493 cm (dark green spot) and .272 cm (very light yellow spot). For green algae, retention values are .77 cm (light yellow spot) and .995 cm (dark green spot). Lastly, cyanobacteria has retention factor .975 cm (dark green spot).
Light grown barely and dark grown barely are grown in different environmental light conditions, which does make an effect on the pigment composition for both barely. Based on the results, both dark and light grown barely have chlorophyll a present as well as a carotenoid. Both plants seem to have similar retention values for the “dark green” spots at the TLC strips. The light grown barely has .9795 cm while the dark grown barely has .90cm. This corresponds to chlorophyll a presence. The retention values of .70 cm (a yellow spot) for the light grown barely and .68 cm (a faded yellow spot) confirms the presence of a carotenoid in both plants. However, both plants are different in the way that light grown barely has more peaks than dark grown barely. Even thou one type of pigment can represent various peaks in a graph, the expression of chlorophylls in light grown barely is greater than that in dark grown barely (3 peaks vs. 1 peak). This may be due to the direct exposure of light to light grown barely. Chlorophyll absorbs red and blue colors in light in turn emitting the color green as a reflection and making the light grown barely greener. Dark grown barely seems to be more yellow due to the lack of chlorophyll abundance and dominance of carotenoids which are accessory pigments and do not emit green color from light absorbance. Therefore, this rejects our null hypothesis that the presence of light exposure does not influence pigment composition of light grown barely and dark grown barely.
Part 2A- 2B
Based on the results, barely does have evolutionary close related relatives. The results show that red algae have four peaks: 2 chlorophyll a, 1 chlorophyll c, and a carotenoid. This is a very similar result to that of light grown barely which had 2 chlorophyll a, one chlorophyll b and a carotenoid. The retention factors are also very similar. The retention spot for red algae however, only shows one dark green spot at .78 which accounts for chlorophyll very close to the chlorophyll in retention .85 for light grown barely. Green algae also expresses two peaks both for chlorophyll a and b along with retention values .995 and .77 which are very similar to the . Retention values for light grown bacteria at .975 and .70. This similarity in retention values means that the pigments that have these retention values have similar but not identical size, or polarity, or molecular weight. More closely related to dark barely is brown alga which has 2 peaks one is chlorophyll and the other a carotenoid. Retention factors however do not match since dark grown barely has .90 cm and .68 while brown algae has retention at .493cm and .272 cm. Cyanobacteria shows three peaks all chlorophylls but no carotenoids, which can have a close relatedness to light grown barely. In general, neither of the organisms had an exact evolutionary identity to barely, but based on the results, red algae seems to be the most closely related to light grown while brown algae to dark light grown algae. Therefore, we reject the null hypothesis that : brown, red, green algae or cyanobacteria pigment each show pigmentation differences compared to barely pigments and are therefore all evolutionary unrelated to barely.
The flaw for both experiments is failure to accurately see with the naked eye the different pigment spots on the TLC strip, which may have made the results more accurate. For example, red algae only had one retention value (for the dark green spot) and ignore the presence of possible faded carotenoids. One solution is to make darker spots on the TLC strips by making 20 instead of 10 repeated spots. This will make the spot much more visual to the naked eye and easier to measure.
Blankenship, R. E. “Early Evolution Of Photosynthesis.” Plant Physiology 154.2 (2010): 434-
438. Academic Search Complete.
Bran, Oana , Camelia Nicula, and Alina Buie. “Accumulation of Chlorophyll and Carotenoid Pigments in Plants Grown under Artificial Light.” Carpathian Journal of Food Science and Technology 4 (2012): 37-45.
Reinbothe, Steffen, and Christiane Reinbothe. “The Regulation of Enzymes Involved In Chlorophyll Biosynthesis.” European Journal of Biochemistry 237.2 (1996): 323-343