Dissecting Photosynthesis Through Emerging Properties in the Chloroplast

Emerson Eggers 002:010:A18 March 5, 2012

1

Abstract

The amount of sugar produced as a byproduct of photosynthesis is greatly

dependent upon multiple variables, which may include illumination, temperature,

and internal factors within the chloroplast (Frankel et al. 101). Photosynthesis is a

very complex process, by which many individual chemical reactions work together

to achieve one goal. Through these experiments you are able to see how

photosynthesis depends on certain components to be present within the

chloroplast, and without these components, photosynthesis is not able to take place.

In order to extricate the effects of these multiple components within a chloroplast in

photosynthesis, different variables were controlled within seven test tubes in order

to determine in which state the chloroplast is most effective in carrying out

photosynthesis (Frankel et al. 103).

Introduction

Photosynthesis can be defined as the plants ability to capture light energy

from the sun, and convert it into chemical energy used by the plant to perform many

functions (Campbell et al. 188). Either directly or indirectly, photosynthesis is the

basis for the sustenance of life on earth, as every organism, whether it’s an

autotroph, heterotroph, or phototroph takes part in the process of photosynthesis

(Campbell et al. 185). Photosynthesis is a complex process, in which at each level

there are emerging properties that become evident. At the first level, is the leaf,

which is the primary source of photosynthesis in the plant, although the stem, and

anything else green are also sites of photosynthesis. The leaf’s shape contours to the

2

needs of the plant, and its structure is designed to maximize the amount of light the

plant will be able to take in (Campbell et al. 187). Within the interior tissue of the

leaf are the mesophyll cells, in which the chloroplasts are located (Campbell et al.

186). The chloroplasts are the sites of photosynthesis, as the chlorophyll pigment

located within the chloroplasts, is the component in which absorbs the light energy

in order to drives the synthesis of compounds (Campbell et al. 186). Although from

a broad perspective it sounds like a relatively simple process, it is the chemical

reactions within these chloroplasts, in which were most important to us during our

experiments, as they were the deciding factor in the rate of synthesis.

Although the higher levels of properties were mentioned above, our main

interest throughout this laboratory was the function of chloroplasts in the process

of photosynthesis. We have already learned that they are the sites of

photosynthesis in the plant, but our questions during these experiments are what

factors surrounding chloroplasts play a role in the production of sugar. When

examining a chloroplast, it can be broken down into two separate, but

interdependent reactions each occurring in a different region of the chloroplast.

The reactions in which takes place first are the light reactions, which take place

inside the thylakoid membrane (Campbell et al 187). During these reactions, light

energy is changed into chemical energy in the form of ATP and NADPH, later used to

drive the Calvin Cycle (Frankel et al. 101). The process of transforming this light

energy into chemical compounds occurs through two systems, photosystem II and

photosystem I, ordered by the way in which they occur within light reactions

(Campbell et al. 194). The process of bringing in light and water molecules into a

3

photosystem and transforming them into electrons, which will then go through

many chemical reactions is one of the most important processes of photosynthesis,

and the basis for all of our experiments. Within this process all of the variables

mentioned before will be precedent. The second reactions in photosynthesis are the

Calvin Cycle Reactions. Within these reactions, carbon dioxide is synthesized into

sugars with the help of the byproducts ATP and NADPH from the earlier light

reactions (Frankel et al. 101). This process has three phases to it; which include

carbon fixation, reduction, and regeneration.

In order for photosynthesis to be carried out, many things must all be

properly functioning within the chloroplast. However, as one can assume, no

process is always smooth. Through the work in experiment 1 we were able to show

how illumination, temperature, and internal factors such as a diuron affected a

chloroplast during photosynthesis. In order to thoroughly understand

photosynthesis and our associated experiments I have devised some hypotheses in

which will allow us to thoroughly understand what we are testing.

Hypothesis 1: Without adequate illumination, photosynthesis will not occur.

Hypothesis 2: By boiling a chloroplast, it will affect the rate of decolorization.

Hypothesis 3: With the presence of diuron, photosynthesis should not take place.

Hypothesis 4: In a chloroplast with a DCPIP electron acceptor, a phosphate buffer,

and a chilled chloroplast suspension B, with no other variables, photosynthesis will

occur.

Hypothesis 5: Around the absorption spectrum of red light (620) and blue light

(420), will be the highest absorption rates from the photosynthetic pigments.

4

Hypothesis 6: Using chromatography, we can separate photosynthetic pigments

into different band orders, based on polarity.

Materials and Methods

* Procedures were obtained through the Principles of Biology Lab Manual*

In experiment 1, we sought to examine the affects of different variables on

the process of photosynthesis. In this experiment, the essential product was

spinach chloroplasts, which were made by grinding spinach leaves down in order to

then isolate their chloroplasts (Frankel et al. 104). Once filtrated through

cheesecloth with buffer, these samples were then diluted, chilled and inverted in

order to get the final product, which was chloroplast suspension B. This suspension

was then placed into 6 different test tubes at increments of 1ml and 2ml. Although

the spinach was our main ingredient, which was put into every test tube except tube

number 5, each tube was unique in its own way. Along with the spinach solution, a

phosphate buffer was also placed into every tube. Tubes 1 and 3 were our

controlled tubes in which only contained chloroplast suspension B and the

phosphate buffer, while the others all had another substance in them whether it was

diuron, DCPIP, or aluminum foil.

By looking at the experiment in which involves the effect of illumination on

the chloroplast, the first hypothesis can be illustrated clearly. The procedure for

determining the affect of illumination is very simple and was able to be carried out

in solely test tube 7. This experiment involved putting 3ml of the phosphate buffer,

2ml of chilled chloroplast suspension B, and 20 drops of DCPIP inside the test tube,

5

while wrapping aluminum foil around the outside of it, as a way to isolate the

components from the light (Frankel et al. 103). Once made, resuspend the

chloroplasts by inverting the tube using parafilm paper 2-3 times, and set into the

illumination apparatus with the light on (Frankel et al. 103). Observe the changes

within the tube in intervals of 2 minutes, as the DCPIP should change colors as it

becomes reduced by oxygen. After 20 minutes, you should have your final results.

To consider the affect of boiling a chloroplast on the chloroplast’s rate of

decolorization in hypothesis 2, we looked at the experiment involving test tube 5 in

which 2ml of boiled chloroplast suspension B was added in place of the regular

chilled chloroplast suspension B solution. This allowed us to isolate the effects of

boiling a chloroplast in comparison to its rate of decolorization. In this experiment

we put 3ml of the phosphate buffer, 2ml of the boiled chloroplast suspension B, and

20 drops of DCPIP (Frankel et al 103). Remember not to put any of the chilled

chloroplast suspension B into the tube, as we want to isolate the effects of the boiled

chloroplast. Once prepared remember to resuspend the tube by inverting it with

parafilm paper again and proceeding to immediately put it into the illumination

apparatus. Since it contains an artificial electron acceptor any changes in the

chloroplast will be able to be observed through a change in color (Frankel et al.

101).

In experiment 1, we sought to measure the effects of diuron on the rate of

reduction for DCPIP. Diuron is a pesticide in which functions in plants by inhibiting

the Hill Reaction in photosynthesis, preventing carbon dioxide fixation, and limiting

the production of ATP (Hess and Warren, 2002). It is harmful to plants as both of

6

those are used for metabolic processes within the plant, and then eventually diuron

will stop all growth within the plant (Hess and Warren, 2002). Through the use of

diuron in this experiment we should be able to observe the outcome for hypothesis

3. For this experiment we used 3ml of phosphate buffer, 2ml of chilled chloroplast

suspension B, 20 drops of DCPIP, and 2 drops of the diuron solution (Frankel et al.

103). Once again, once the solution is prepared you resuspended the solution and

stuck it into the illumination apparatus checking it every 2 minutes for a period of

20 minutes to observe changes within the solution.

In experiment 1, we are also trying to determine if photosynthesis will occur

in a solution in which only contains the phosphate buffer, chilled chloroplast

suspension B, and DCPIP. To examine if this will happen we set up two test tubes in

which were the controlled variables. In tube number 2 we added 4ml of the

phosphate buffer, 1ml of the chloroplast suspension B, and 20 drops of DCPIP

(Frankel et al. 103). While in tube number 4 we added 3ml of the phosphate buffer,

2 ml of the chilled chloroplast suspension B, and 20ml of DCPIP (Frankel et al. 103).

Repeat the final steps of the process and observe results.

*Contains acetone, please use gloves and do work under fume hood*

As for experiment 2, it was performed in order to determine which pigments

on the chloroplast have the highest absorbance rate going along with hypothesis 5,

or in other words which pigments contribute the most to the photosynthetic process

(Frankel et al. 105). To perform this experiment you will start by separating the

pigments from the leaf using 100% Acetone with 2g of tore up leaves. Once a

homogenous mixture, pour contents into a funnel containing glass-wool, and into a

7

13mm test tube (Frankel et al. 105). Making sure to not look at the ultraviolet light,

compare the newly made solution, with the solution made from experiment 1. In

order for the Spectronic 20 spectrophotometer to read your solution, it must be

diluted. To do this pour the 80% acetone into your test tube until it’s about 3cm

from the brim. If it now falls within 0.3-0.5 O.D. at 663nm your sample is ready to

be used, if not dilute again Frankel et al. 105). Now you can record the absorbance

rates from 390nm-690nm in increments of 20nm, in order to see the optimal

absorbance number for plants.

*Experiment contains petroleum ether: acetone, 9:1, volatile and flammable*

Chromatography is used in order to separate molecules based on their

relative polarities (Frankel et al. 106). Through the use of chromatography, we can

describe my 6th and final hypothesis. Knowing that their polarities are based on the

number of polar oxygen’s in the molecule in relation to the polarities of the solvent

and water on the paper, it is relatively simple to predict bond order (Frankel et al.

106). First, start out by drawing a pencil mark 2cm from one end of a strip of 3MM

chromatography paper. Next you are going to want to take a bit of the leftover

green leaf extract from experiment 2, and make a streak along that line you just

drew. In order to make sure it will show up later in the experiment, repeat the

streak 10 times taking short breaks in between each streak. Next, stick into a test

tube containing 2ml of the solvent (petroleum ether: acetone, 9:1), and recap the

tube (Frankel et al. 106). After about 10 minutes you should have your results.

Results

8

The results that were obtained from experiment 1 involving the rate of

DCPIP reduction in the chloroplast are shown below.

Effect of Chloroplast in PhotosynthesisTube Room Temp

PO4 Buffer Chilled ChloroplastSuspension B

BoiledChloroplastSuspension B

Diuron(drops)

DCPIP(drops)

Color of Solution at To

Results (color at end)

1 5ml 1ml 0 0 0 Green Green (due to chloroplasts)

2 4ml 1ml 0 0 20 drops Blue Green (photosynthesis occurs)

3 4ml 2ml 0 0 0 Green Green (due to chloroplasts)

4 3ml 2ml 0 0 20 drops Blue Green (photosynthesis occurs)

5 3ml 0 2ml 0 20 drops Blue Blue (Photosynthesis does not occur)

6 3ml 2ml 0 2 drops 20 drops Blue Blue (contains herbicide)

7 3ml 2ml 0 0 20 drops Blue Blue (light reactions did not occur)

(Frankel et al. 103)

The results from experiment 2 on the absorbance rates at different

wavelengths are listed below.

Absorption Rates at Varying WavelengthsWavelengthIn nm

FirstReading (663)

390 410 430 450 47

0

490 510 530 550 570 590 610 630 650 670 690

O.D. Units .42 .85 .90 1.3 1.1 .70 .29 .07 .05 .06 .1 .12 .14 .16 .34 .41 .07

(Frankel et al. 106)

*Some results may differ from the norm in Experiment 2 due to our Spectronic 20 spectrophotometer not properly working during some tests*

9

From experiment 3 we were able to predict bond order based upon a

molecules relative polarities. Our results for bond order are stated below.

Separating Pigments

Characteristics of Pigments Carotenes Orange with no

oxygenXanthophylls Yellow with 2

polar oxygen’sChlorophyll a Bluish-green

with 5 polar oxygen’s

Chlorophyll b Yellow-green with 6 polar oxygen’s

(Frankel et al. 106) (Frankel et al. 106)

Discussion

My first four hypotheses are all based off of experiment 1, in which we were

trying to measure the rate of DCPIP reduction in a chloroplast. We were able to do

this by isolating different variables within 7 different test tubes, including 2 control

groups, which were tubes 1 and 3. In order to then see if the DCPIP electron

acceptor was reduced we had to put it into an illumination apparatus for 20

minutes, continually checking it in 2-minute intervals. In its oxidized state, DCPIP is

a dark blue color, but as it becomes reduced, that blue will continually fade until it

becomes clear (Frankel et al. 101). In experiment 1, when determining whether

photosynthesis will happen in a certain solution, it is this final color change in which

will give us the answer.

In my first hypothesis I stated that without adequate illumination,

photosynthesis would not occur, as the DCPIP would not be reduced. In order to

10

Bond OrderPhotosynthetic Pigment

Highest Carotenes

Next Xanthophylls

Next Chlorophyll a

Lowest Chlorophyll b

support this, test tube 7 was wrapped in aluminum foil, which prevented light from

reaching the otherwise normal solution. From the results I obtained after 20

minutes of illumination, my hypothesis was in fact correct, as the DCPIP remained in

its oxidized blue state by the end. The process by which light is taken into the

pigment molecules in a photosystem is vitally important to photosynthesis, as it is

the first process by which the rest of the rest of the process of photosynthesis

depends on. Without this light, a plant is forced to make less organic compounds

that are vital to its survival. If the plant goes through extended periods without

light, it will most likely die, as it would have then exhausted all of its food reserves

(Campbell et al. 187).

In my second hypothesis, I stated that through the process of boiling a

chloroplast, it would then affect the rate of decolorization. This was presented

through test tube 5 in which contained 2ml of boiled chloroplast suspension B, a

phosphate buffer, and 20 drops of DCPIP. The results from this experiment indicate

that boiling a chloroplast did indeed have a great affect on the rate of decolorization.

However, that is not saying that the DCPIP changed to a clear substance, but the

contrary, by actually staying dark blue. The reason for the omission of

photosynthesis in this experiment is due to the effects that boiling has on the

chloroplast. When boiled, the 3-D structure of the chloroplast changes, which in

turn leads to DCPIP not being able to be reduced as it does not the enzymes it needs

in order to reduce (lab discussion). However, there is one thing that I thought

would be interesting to test in order to thoroughly rule out any other possibilities to

why photosynthesis did not occur in this reaction. My idea would be to repeat this

11

experiment, except this time test the theory of a boiling chloroplast at varying

temperatures. This could show us if a chloroplast is ineffective in the process of

photosynthesis at every temperature, or if as you get closer to room temperature

photosynthesis becomes a little more prevalent in the solution.

As for my third hypothesis, I declared that with the presence of diuron,

photosynthesis would not take place. Once again my results from tube 6 showed

that my hypothesis is true, in that if a diuron is present in the solution, the light

reactions are unable to take place, ultimately stopping photosynthesis. A diuron in

effect causes a blockage during the electron transport chain from photosystem II to

photosystem I (Campbell et al. 195). Since the plant is no longer able to get

electrons across this chain and through the cytochrome complex, it is also then

unable to produce NADPH and ATP, which are vital aspects for the Calvin Cycle to

work. Over time some plants have evolved to minimize the effects of a diuron.

These plants use what is called cyclic electron flow, which is when a plant will only

use photosystem I in the light reactions (Campbell et al. 195). This is done when

Ferredoxin returns electrons back into the cycle where they go back through the

process once again (Campbell et al. 195). Although this process seems ideal by even

allowing for production of ATP, it does not allow for the production of NADPH,

which is an essential item in the following reactions (Campbell et al. 195).

Following along into my fourth hypothesis, we will now encounter the two

items in which we initially expected to produce photosynthesis in the experiment,

which are the test tubes 2 and 4. In this hypothesis I stated that in a chilled

chloroplast suspension B with the phosphate buffer and DCPIP, photosynthesis

12

would occur. As expected this was proven true as one might expect. No outside

variable were used in this experiment, so we knew that it would act as any normal

plant does during photosynthesis, as the DCPIP has the same structure as a regular

electron acceptor, allowing for a smooth experiment.

We will now examine my fifth hypothesis in which will take us into

experiment 2. Through this hypothesis I stated that around the absorption

spectrums of red light (620-700) and blue light (420), would be the greatest

absorption rates for our photosynthetic pigments. Although in this experiment it

was harder to find definitive results, based upon absorption rates in the vicinity of

these two wavelengths, it is safe to say that the results supported this hypothesis.

To clearly explain that, you can look at the absorption rate at 430nm, which is very

close to blue, and possessing a 1.3 O.D.; the highest of any value we obtained. This is

significant, as plants are selectively permeable, and will only take in certain

wavelengths to be used as energy, while shielding any potentially harmful rays from

the plant (Campbell et al. 190). Since all of the rays coming in are good for the plant,

it allows for the plant to effectively begin the process of photosynthesis.

In my last hypothesis, I hypothesized that through the use of

chromatography, we can separate photosynthetic pigments into different orders

solely based upon polarity. Through the use of chromatography paper, leaf extract,

and a solvent, we were able to obtain results in which proved my hypothesis

correct. After 10 minutes of waiting, there were clearly 4 individual dots from each

of the pigments. This is significant in the process of photosynthesis because it

shows us how even the different pigments within the chloroplasts all correspond to

13

different duties. While pigments such as chlorophyll a and b help with the transfer

of the light energy into the reaction center complex, carotenes and xanthophylls can

help protect the chlorophyll from dangers such as excessive sunlight (Kimball, John.

1994).

In the biological world there are new properties emerging at each level, and

photosynthesis is no exception (Campbell et al. 3). Photosynthesis involves the

specific interaction of components in a chloroplast (Campbell et al. 3). An example I

am going to use is a factory assembly line, in which each worker has a specific job in

which they must do, before that item is passed down to the next worker. The

assembly line will start with raw material (in the case of photosynthesis this is the

sun and water), and as it works its way down the line, some things may be added or

taken away from it in order to transform it into its final product. Photosynthesis

works this way too, as the sun and water are necessary for the first light reactions to

occur. Once the light reactions occur, their product, which is ATP and NADPH, is

then transferred to the Calvin Cycle in order to fuel the reactions between the break

down of carbon dioxide into sugar. However, if for some reason this whole process

encounters a problem such as a diuron during the light reactions, the whole process

of photosynthesis could be affected. So the overall point is that the structure of a

chloroplast is designed in order to have different functions at each level, and all of

these functions work as a team in order to keep not only the chloroplast in good

shape, but the plant as well.

References

(1) Campbell, N. and Reece, J. (2008) Biology: Eighth Edition. Pearson Education Inc., San Francisco

14

(2) Frankel, J., Williams, N., Irish, E., and Stefaniak, J. (2011). Principles of Biology I

Laboratory Manual: Seventh Edition. Pearson Education Inc., Boston, MA. “Three experiments on Photosynthesis,” pp.100-107.

(3) Hess, D., Warren, F. (2002). “The Herbicide Handbook of the Weed Science

Society of America: Eighth Edition.” pp. 159-161. [Accessed on 3/5/12].

Environmental Monitoring Branch, Department of Pesticide Regulation. “Environmental Fate of Diuron.” www.cdpr.ca.gov/docs/emom/pubs/fatememo/diuron.pdf

(4) Kimball, John. (Last published book, 1994). “Chlorophylls and Carotenoids.”

[Accessed 3/5/12]. www.users.ren.com/jkimball.ma.ultranet/BiologyPages/C/chlorophyll.html

** Lab partner: Colleen Peters

15