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2Molecular Phylogeny of the Philippine endemic genus Antherostele Bremek. (Rubiaceae) inferred from ITS data (nrDNA) and its conservation status

Photosynthesis

PhotosynthesisJohn Benedict C. Palo, Kyra Christine A. Rios, Rochelle Anne R. Tan, Nathea Agatha C. Valdez

Submitted to: Maam Elena RagragioSubmitted: September 20, 2012ABSTRACTThis activity explores the different factors and processes that affect the photosynthetic rate of plants. Chlorophyll, light, and carbon dioxide are the important things in photosynthesis since chlorophyll is the necessary green pigment, light provides the energy needed, and carbon dioxide combines with ribulose biphosphate (RuBP) to make its food (simple sugars). Because plants only use the hydrogen in the water it absorbs, oxygen is released back to the atmosphere; hence oxygen liberation. Chloroplast pigment separation is important in determining which wavelengths plant need from the sun in order to drive photosynthesis.1. Introduction Photosynthesis is an important process that benefits all living things. If it were to suddenly stop because of an unexpected reason, a chain of unfortunate events is to happen; therefore, we must look closer to further understand what and how factors affect photosynthesis.The group hypothesized several things. First, the amount of chlorophyll, light and CO2 present and absorbed by the plant is directly proportional to its photosynthetic rate. Second, the oxygen released by the plant is the oxygen from H2O. Lastly, the pigment nearest the top of the paper is the most soluble.

By the end of this activity, this group is expected to: determine the role of chlorophyll, light, and carbon dioxide in photosynthesis, observe oxygen liberation during photosynthesis, and demonstrate how chloroplast pigments are separated and to identify these pigments.2. Methodology2.1. The Role of Chlorophyll in PhotosynthesisA variegated leaf was sketched that showed its pattern of green and white areas. The leaf was then boiled in water for 10 minutes then immersed in a test tube with 95% ethyl alcohol. Afterwards, it was placed in a hot water bath until pigments were extracted then it was washed with water and tested for presence of starch using IKI solution.2.2. The Role of Light in PhotosynthesisAfter a potted plant was placed in the dark for 48 hours, random leaves were chosen to wrap portions of it with carbon paper. The plant was then exposed to light for 5 hours and tested for starch as previously done in 2.1.

2.3. The Role of CO2 in Photosynthesis

Three test tubes labeled A, B, and C were filled with previously boiled then cooled distilled or tap water. Hydrilla sprigs were placed invertedly into tubes A and B and a pinch of NaHCO3 was added to tubes B and C. The 3 tubes were then exposed to bright light then the group determined whether bubble evolution occurs in A and/or B. After the hydrilla sprigs were removed, 5 drops of phenol red were added to each then gently shook (phenol red changes to yellow at low pH). The color intensities of the solutions in the test tubes were then compared.2.4. Oxygen Liberation in Photosynthesis

Once the 1L beaker was filled with tap water, air was gently blown into it using a plastic straw for 3-5 minutes to enrich CO2. Afterwards, the ends of the hydrilla sprigs were cut under water (make sure the cuts face the tube of the funnel) and a thistle funnel was inserted over those. A test tube filled with water was place upside down over the tube of the funnel which was partially dipped in the water of the beaker. The emerging bubbles coming out from the cut ends were then observed and the nature of the gas was determined by placing a lighted match in the test tube.2.5. Separation of Chloroplast Pigments

The leaf extract was prepared by getting 15 to 20 mature hibiscus leaves, cutting it into small pieces, then putting it in a vial, and setting it aside. The filter paper was prepared by fitting a piece of paper into a clean, dry test tube and setting this as the mold. With this, cut the filter paper into strip, all the while making sure that only the edges will be held since fingerprints on the surface of the paper may affect chromatogram. When the filter paper can slide in and out of the tube without touching the side, a 2 cm mark from the top and a 1cm mark from the base of the strip was made using a pencil. A point was then cut at the base of the strip. Mark the outside of the test tube to show proper solvent level. Remove the paper strip from the dry test tube and lat it on the table. Meanwhile, solvent was poured into the dry test tube up to the marked line. The cork was replaced without the paper strip and kept inside.

The chromatogram was prepared by dipping capillary tube or a fine-tip dropper into the vial which contained the extract. The drop that came out of the capillary tube was placed onto the center of the 1 cm mark at the base of the tip and dry by waving paper in the air. This process was repeated until the spot became fully saturated with the green extract (18 times). The green spot was as small as possible and the drops were added only after each drop had completely dried up. The end result was a paper with a small, dark green spot in the center of the 1 cm mark with the spot not reaching the sides of the strip.

After the spot has completely dried up, the cork was removed from the tube and the paper strip was placed on the hook and inserted into the test tube containing the solvent mixture of 95 parts petroleum ether and 5 parts acetone with the solvent level below the 1 cm mark. Minor adjustments were made with the paper clip. The cork was replaced with the strip and the flow of the solvent was observed until the 2 cm mark. The paper was then removed from the tube and left to dry. After which, the chromatogram showed the different pigments present in the specimen.3. Results

3.1. The Role of Chlorophyll in PhotosynthesisStarch was present in the whole leaf. 3.2. The Role of Light in Photosynthesis

Starch was only present in the areas uncovered by carbon paper.

3.3. The Role of CO2 in Photosynthesis

Bubbles were formed in both test tubes A and B although tube B produced more. Table 1. Formation of BubblesTest TubeColor Intensity

A+

B++

C+++

3.4. Oxygen Liberation in Photosynthesis

Bubbles emerged from the cut ends.3.5. Separation of Chloroplast Pigments

From top to bottom, the pigments were carotene (orange), xantophyll (yellow), chlorophyll a (blue green), and chlorophyll b (light green).4. Discussion

4.1. The Role of Chlorophyll in PhotosynthesisThe leaf was boiled so as to burst the chloroplast, making it easier to extract chlorophyll.

The green areas are photosynthetic whereas the white ones are non-photosynthetic. Based on the experiment, only those areas with chlorophyll (green areas) can undergo photosynthesis.4.2. The Role of Light in PhotosynthesisIt was necessary so as to use up the plants starch reserves. Only the parts that were uncovered showed positive reaction to the starch test. It can be concluded therefore that light is necessary for photosynthesis since only the uncovered parts were able to produce starch.4.3. The Role of CO2 in Photosynthesis

Test tube C had the most color intensity followed by B, then A upon addition of phenol red. Bubbles were formed in tubes A and B since the NaHCO3 supplied more CO2, hence tube B produced more bubbles since it had both a sprig and NaHCO3. Boiled water was used so as to eliminate dissolved air.

4.4. Oxygen Liberation in Photosynthesis

The tube was placed upside down so as to probably, the sprig shall catch all the water test tubes got to bring. The bubbles indicate carbon dioxide mixing with water, and releasing oxygen.

4.5. Separation of Chloroplast Pigments

The colors present were orange which represents carotene, yellow which represents xantophyll, blue green for chlorophyll a, and light green for chlorophyll b. The least soluble is chlorophyll b since this is at the bottom and therefore took the longest amount of time travelling up the paper.References[1] Committee on Biology 21, Biology 21 General Botany Laboratory Manual Vol. 2008-2009, Exercise 13: Respiration

[2] James D. Mauseth, Botany: An Introduction to Plant Biology, Jones and Bartlett, Fourth Edition, pp. 244-246

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