BIOL 109L Laboratory Eight Fall 2018 Plants and How They Feed Us – Part II

BIOL 109L Laboratory eight fall 2018 Plants and how they feed us – part II

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Introduction: Photosynthesis is one of the most important biochemical processes in plants and is amongst the most expensive biochemical processes in plant in terms of investment. Photosynthesis is also the major biochemical process that has driven plant form and function. Photosynthetic organisms use solar energy to synthesize carbon compounds that cannot be formed without the input of energy. More specifically, light energy drives the synthesis of carbohydrates from carbon dioxide and water with the generation of oxygen (Bryant et al., 2006). In addition to the chlorophyll pigments, there are other pigments present. During the fall, the green chlorophyll pigments are greatly reduced revealing the other pigments, such as Carotenoids that are pigments that are either red, orange, or yellow. Chlorophylls degrade into colorless non-fluorescent chlorophyll catabolites (NCCs). As the chlorophylls degrade, the hidden pigments of Carotenoids are revealed in a process called Photoprotection: Carotenoids protect the leaf against the harmful effects of light and low temps. By shielding the leaf with Carotenoids, the plant manages to reabsorb nutrients (especially nitrogen) more efficiently, including the components of chlorophyll. These are stored within the phloem for the next growing season (Hortensteiner, 2006).

Objectives: The purpose of this lab is to study the relative abundance of photosynthetic pigments in plant leaves before and after chlorophyll degradation has occurred. You will: I) Extract pigments from living and dead plant material to determine total chlorophyll content. II) Determine chlorophyll A:B ratio and total content of chlorophyll A, chlorophyll B, -carotene, phycoerythrobilin, and phycocyanobilin. III) Understand exactly what knowledge of the relative amounts of these pigments can inform you about the general health and wellbeing of a plant. IV) Learn and understand how to carry out a statistical and graphical analysis, and an appreciation of its sources of error and uncertainty. V) Turn your photosynthesis working knowledge up to an eleven!

Frist off: weigh plant samples: In part I, you weighed out two grams of each plant sample and add 20 ml absolute ethanol and store in the dark at -20°C for the next lab section. Re-record the exact weight to three significant figures of each sample in the table below.

• Remove and KEEP the (now green) ethanol –it contains the chlorophyll molecules that you need for this lab. Place the ethanol from each sample into a clean, separate, clearly labeled 50 ml corex tube.

• Weigh the plant material following chlorophyll extraction and record the exact weight to three significant figures of each sample in table one.

• Place plant samples back in corex tubes at place in incubator at until next week. Then weigh and record the exact weight to three significant figures of each sample in the table below.

Table 1. Plant leaf sample name and exact dry weight Sample name Exact wet weight Exact wet weight Weight difference Exact dry weight of before Ethanol after Ethanol after Ethanol plant material after extraction (Grams) extraction (Grams) extraction (Grams) one week at 60ºC from last week (Grams) Dead leaves

Flaccid leaves Fresh leaves

The Chlorophyll "A/B ratio" - a unitless value that is used to estimate how much photosystem I vs. photosystem II is in the chloroplasts of leaves.

In higher plants, there are two species of chlorophyll molecules, Chlorophyll A and Chlorophyll B. These two species of chlorophylls have very similar spectral qualities in that they absorb red and blue light but do not absorb green light. They differ in that the exact or peak wavelength of red and blue light they absorb:

Chlorophyll A Absorbs red light maximally at a wavelength of 664 nanometers. Chlorophyll B Absorbs red light maximally at a wavelength of 667 nanometers. B Carotene Absorbs at 450 and 520 nanometers. Phycoerythrobilin Absorbs maximally at a wavelength of 565 nanometers. Phycocyanobilin Absorbs maximally at a wavelength of 621 nanometers.

Measuring the relative amount of chlorophyll A and B in a leaf: In plants, there are only two species of chlorophyll molecules, Chlorophyll A and Chlorophyll B. A useful measure of photosynthetic status in plants is the relative amount of Chlorophyll A versus Chlorophyll B, referred to as the "Chlorophyll A/B ratio". It is used as an indicator of the how leaves adapt to the level of light they are grown under. It is an indirect indicator of how much of two different kind of photosystems it has synthesized in the chloroplast for trapping light for photosynthesis.

What is a photosystem? Chlorophylls are imbedded in one of two huge chloroplast membrane light harvesting protein complexes called photosystems, referred to as photosystem I and photosystem II. Both photosystems cooperate with each other in turning light energy into biochemical energy. They differ in the number of the two kinds of chlorophyll molecules they contain. For example, Photosystem I has 4 times more Chlorophyll A than Chlorophyll B, while Photosystem II has about the same amount of Chlorophyll A to B:

Figure 1: A few photosynthetic pigments. The curves in Photosystem I has ~ 40 Chlorophyll B this graph show the efficiency at which each pigment molecules and 160 molecules Chlorophyll A absorbs the different wavelengths of visible light. Line color indicates the pigment’s characteristic color. Using a combination of pigments allows photosynthetic Photosystem II has ~ 100 Chlorophyll B organisms to maximize the range of wavelengths they molecules and 100 molecules Chlorophyll A can capture for photosynthesis

The plant alters the number of the two different kind of photosystems it uses depending on the level of light the leaf receives, such as a leaf positioned in direct high sunlight versus one that is shaded. When a leaf is positioned in direct sunlight, it needs more PS I to fully convert the abundant light energy into biochemical energy. Typically, leaves adapted to full sunlight synthesize more PSI and will therefore have a larger number for the Chlorophyll A:B ratio than shade leaves. Alternatively, light starved leaves need more PS II to convert what little light energy there is into biochemical energy. Thus, shade leaves have lower Chlorophyll A:B ratios, e.g., 2:1.

Data collection: So, you will need to collect absorbance readings at the following wavelengths: AB 450 nanometers Max B Carotine AB 520 nanometers Max B Carotine AB 565 nanometers Max Phycoerythrobilin AB 621 nanometers Max Phycocyanobilin AB 649 nanometers For Chl A:B ratio AB 664 nanometers Max ChlA AB 665 nanometers For Chl A/B ratio AB 667 nanometers Max Chl B

• Work through each wavelength in the order shown above

• Each time, set the wavelength (nm) accordingly of the spectrophotometer and blank with 1 ml of absolute ethanol. Read 1 ml of all three samples dead leaves, flaccid leaves and fresh leaves). Every time you change nm, re blank to zero with ethanol.

• Repeat the reading three times for each wavelength and record the readings on table two below. EVERY group member need to fill in their tables.

Table2: All absorbance wavelength readings to determine the group mean values Sample Ab 450 Ab 520 Ab 565 Ab 621 Ab 649 Ab 664 Ab 665 Ab 667 Dead leaves (1) (2) (3) mean Flaccid leaves (1) (2) (3) mean Fresh leaves (1) (2) (3) mean

Sail forth - steer for the analysis of the data. Reckless O soul, exploring:(badly paraphrased from Whitman, 1872). • Insert your groups mean values for each absorbance setting for dead, flaccid, and fresh leaves in tables three, four, and five respectively. Collect the mean values for each absorbance setting from the other four groups and insert them into each corresponding table.

• Calculate the average absorbance value Mean (x̅ ) for each absorbance setting for dead, flaccid, and fresh leaves in tables three, four, and five respectively.

• Calculate the Difference from the mean (X-x̅ ) for each absorbance setting for dead, flaccid, and fresh leaves in tables three, four, and five respectively.

• Calculate the (Difference from mean)2 (X-x̅ )2 for each absorbance setting for dead, flaccid, and fresh leaves in tables three, four, and five respectively.

• Calculate the sun (∑) of the (Difference from the mean)2 ∑(X-x̅ )2 for each absorbance setting for dead, flaccid, and fresh leaves in tables three, four, and five respectively.

• Calculate the Variance. The term variance was first introduced by Sir Ronald Aylmer Fisher, FRS (1918), and is a measurement of how far a data set are spread out from their average value. This is the Sum of (Difference from the Mean)2 divided by the degrees of freedom (n – 1). So: ∑(X-x̅ )2 (n-1) Do this for each absorbance setting for dead, flaccid, and fresh leaves in tables three, four, and five respectively.

• Now onto the final step: Determine the standard deviation. This is a measure that is used to quantify the amount of variation or dispersion of a set of data values. A low standard deviation indicates that the data points tend to be close to the mean (also called the expected value) of the set, while a high standard deviation indicates that the data points are spread out over a wider range of values. (Bland and Altman, 1996). The standard deviation is calculated as the square root of the variance (the value you ∑(풙−풙̅)ퟐ just calculated). So: √ = (풏−ퟏ)

Do this for each absorbance setting for dead, flaccid, and fresh leaves in tables three, four, and five respectively.

Table 3: Absorbance values for class groups dead leaf samples Sample Ab 450 Ab 520 Ab 565 Ab 621 Ab 649 Ab 664 Ab 665 Ab 667 Dead leaves (1) (2) (3) (4) (5) Mean (x)̅ 1) 1) 1) 1) 1) 1) 1) 1) (X-x̅ ) 2) 2) 2) 2) 2) 2) 2) 2) 3) 3) 3) 3) 3) 3) 3) 3) 4) 4) 4) 4) 4) 4) 4) 4) 5) 5) 5) 5) 5) 5) 5) 5) 1) 1) 1) 1) 1) 1) 1) 1) (X-x̅ )2 2) 2) 2) 2) 2) 2) 2) 2) 3) 3) 3) 3) 3) 3) 3) 3) 4) 4) 4) 4) 4) 4) 4) 4) 5) 5) 5) 5) 5) 5) 5) 5) ∑(X-x̅ )2

∑(X-x̅ )2 (n-1)

∑(풙 − 풙̅)ퟐ √ (풏 − ퟏ)

Table 4: Absorbance values for class groups flaccid leaf samples Sample Ab 450 Ab 520 Ab 565 Ab 621 Ab 649 Ab 664 Ab 665 Ab 667 Flaccid leaves (1) (2) (3) (4) (5) Mean (x)̅ 1) 1) 1) 1) 1) 1) 1) 1) (X-x̅ ) 2) 2) 2) 2) 2) 2) 2) 2) 3) 3) 3) 3) 3) 3) 3) 3) 4) 4) 4) 4) 4) 4) 4) 4) 5) 5) 5) 5) 5) 5) 5) 5) 1) 1) 1) 1) 1) 1) 1) 1) (X-x̅ )2 2) 2) 2) 2) 2) 2) 2) 2) 3) 3) 3) 3) 3) 3) 3) 3) 4) 4) 4) 4) 4) 4) 4) 4) 5) 5) 5) 5) 5) 5) 5) 5) ∑(X-x̅ )2

∑(X-x̅ )2 (n-1)

∑(풙 − 풙̅)ퟐ √ (풏 − ퟏ)

Table 5: Absorbance values for class groups fresh leaf samples Sample Ab 450 Ab 520 Ab 565 Ab 621 Ab 649 Ab 664 Ab 665 Ab 667 Fresh leaves (1) (2) (3) (4) (5) Mean (x)̅ 1) 1) 1) 1) 1) 1) 1) 1) (X-x̅ ) 2) 2) 2) 2) 2) 2) 2) 2) 3) 3) 3) 3) 3) 3) 3) 3) 4) 4) 4) 4) 4) 4) 4) 4) 5) 5) 5) 5) 5) 5) 5) 5) 1) 1) 1) 1) 1) 1) 1) 1) (X-x̅ )2 2) 2) 2) 2) 2) 2) 2) 2) 3) 3) 3) 3) 3) 3) 3) 3) 4) 4) 4) 4) 4) 4) 4) 4) 5) 5) 5) 5) 5) 5) 5) 5) ∑(X-x̅ )2

∑(X-x̅ )2 (n-1)

∑(풙 − 풙̅)ퟐ √ (풏 − ퟏ)

Results: So, to present this section of the experimental data, fill in table six with the class mean values for all absorbances with the + standard deviation values for each absorbance calculated in tables three, four and five. To this for the dead, flaccid, and fresh leaf samples.

Table 6: Average values of the absorbance of each photosynthetic pigment with + standard deviation values. Sample Ab 450 Ab 520 Ab 565 Ab 621 Ab 649 Ab 664 Ab 665 Ab 667 Dead leaves Flaccid leaves Fresh leaves

Calculation of the chlorophyll A and B ratios: Over the years, plant physiologists have developed what are called empirical formulas for determining the amount of chlorophyll a and chlorophyll b in a mixture of the two. It is empirical because all of its variables (coefficients) are derived by starting with purified Chlorophyll A and purified Chlorophyll B and then mixing the two in various amounts to see how the absorbance at each respective maximal absorbance changes. Fortunately for us, these scientists have done all the hard work for us and all we need to do is determine the absorbance of our leaf chlorophyll solutions at these wavelengths and then plug the values into the empirical formula; (Bryant and Frigaard, 2006).

Thus, insert your class mean absorbance values for dead, flaccid, and fresh leaves in the following equation to obtain this very informative Chlorophyll A:B ratio that you will report in your final analysis:

Chl A = [(-5.2)(A )] + [(13.5)(A )] 649 665 = Chl A:B ratio Chl B = [(22.4)(A649)] + [(-7.07)(A665)]

Equation 1: calculation to determine the chlorophyll A:Chlorophyll B ratio

Record all the Chl A:B ratio values in table seven:

Table 7: Average values of the Chl A:B ratio for dead, flaccid, and fresh leaves. Sample Chl A:B ratio Dead leaves Flaccid leaves Fresh leaves

Conclusions and Questions: FULLY answered all the following questions. In the last lab section of the project you will have time to research the answers online to understand the whole picture that photosynthetic pigments have on plants that feed us.

1) For each leaf sample, describe the relative abundance of each of the five pigments assayed: Dead Leaves Chlorophyll A

Chlorophyll B

B Carotene

Phycoerythrobilin

Phycocyanobilin

Flaccid Leaves Chlorophyll A

Chlorophyll B

B Carotene

Phycoerythrobilin

Phycocyanobilin

Fresh Leaves Chlorophyll A

Chlorophyll B

B Carotene

Phycoerythrobilin

Phycocyanobilin

2) Consider the processes of Photoprotection by Hortensteiner (2006). Fully explain how your data explains this process. Search for a primary literature source to help explain your reasoning and cite it at the end of your explanation.

3) Use the data to explain the significance of the Chl A:B between the three types of leaf tissue. Search for a primary literature source to help explain your reasoning and cite it at the end of your explanation.

4) Time to be honest! Did you get a better understand of why a wee bit of knowledge about how plants work? Do you now understand how to do a statistical analysis? Please let me know what you feel. POINTS WILL NOT BE LOST HERE!!!!!!!!!!!!

O Captain! my Captain! our fearful trip [through plants and how they feed us: part II] is done; The ship has weather'd every rack, the prize we sought is won; (badly paraphrased from Whitman, 1865).

References:

Bland, J.M. and Altman, D.G. (1996). Statistics notes: measurement error. British Medical Journal.312 (7047): 1654.

Bryant DA. and Frigaard NU. (2006). Prokaryotic photosynthesis and phototrophy illuminated. Trends in Microbiology. 14 (11): 488–96.

Fisher, R.A. (1918) The Correlation Between Relatives on the Supposition of Mendelian Inheritance. Translations of the Royal Society of Edinburgh, 52: 399 – 433.

Hortensteiner, S. (2006). Chlorophyll degradation during senescence. Annual Review of Plant Biology. 57: 55–77.

Whitman, W (1865) O Captain! My Captain! In: the pamphlet Sequel to Drum-Taps: When Lilacs Last in the Dooryard Bloom'd and other poems. Washington: Gibson Brothers, pp 3 - 11.

Whitman, W (1872) Passage to India. In: Leaves of Grass. Washington: Gibson Brothers, pp 313 - 323.