MODEL INTRO-MATERIALS AND METHODS PHOTOSYNTHESISAn investigation of the effect of light colour on the rate of photosynthesis (measured indirectly

rate of oxygen production in CAM (represented by Agave americana), C3 (represented by Spinacia oleracea) and C4 (represented by Atriplex canescens) plants evaluated using the leaf

disc floating technique)Research Question: What is the effect of light colour on the rate of oxygen production in CAM, C4 and C5 plants measured through the floating leaf technique?

Background Knowledge:Photosynthesis is a process occurring in all types of plants and some bacteria in which energy from the sun is converted into chemical energy. Later this energy can be released in order to fuel organisms own processes. The light-dependent reactions of photosynthesis produce ATP and NADPH+H+ while light-independent reactions produce carbohydrates, such as glucose. Photosynthesis is one of the only two processes that can convert inorganic carbon into organic.The overall reaction of photosynthesis in most of the plants can expressed in this manner:

6CO2 + 6H2O ----> C6H12O6 + 6O2 sunlightPhotosynthesis takes place in the chloroplasts which are filled with chlorophyll. It

absorbs the light and with its help plants can combine the CO2 with H2O. H2O is absorbed by the roots and delivered to the leaves through xylem vessels, while the CO2 is absorbed by leaves directly from the air. There are tiny pores on the surface of leaves, called stomata, which absorb the CO2, release the O2, a side product of the reaction and act as pipes for releasing water vapor. Photosynthesis can be broken down into two types of reactions: light-dependent and light-independent. In the first one chlorophyll captures energy from the sun and temporarily stores it in form of ATP and NADPH. During the light-independent reaction the energy in those compounds is used to form glucose from carbon dioxide.

Rate of photosynthesis in typical plants can be changed by different factors, such as availability of water and carbon dioxide, and light intensity and color. It can also be affected by the temperature of environment and its pH.

This lab concentrates on the examining possible effect of light color. Light colour is determined by the frequencies of light in its spectrum, which in visible spectrum, ranges from red to violet. Visible light from the sun includes the whole visible spectrum, but not all of it, however, is accepted by the plant. This is why different colors of light may change the (pic. 1) Spectrum of light, accepted by different kinds of chlorophyll

rate of photosynthesis, http://www.uic.edu/classes/bios/bios100/lecturesf04am/lect10.htm

even when illuminated with the same intensity. This happens due to the fact that the chlorophyll can only accept light of certain frequencies. As can be seen on the diagram, chlorophyll cannot accept yellow and green light which gives leaves their typical green “color of life”. The color that is most effectively absorbed by the light is blue.

Leaves contain spaces between their top and bottom layers. This space is a place where gas exchange occurs. The volume is large enough in order to provide enough buoyancy for leaves to float when they are filled with oxygen,

There are three different categories of plants categorized by their type of photosynthesis: C3, C4 and Crassulacean Acid Metabolism (or CAM). C3 are the most common type, used by most of the plants in our environment. Their name carries their most distinguishing characteristic: the type of compound that the CO2 is incorporated in before it is used in photosynthesis. In C3 it is stored as a 3-carbon chain, in C4 as a 4-carbon chain and in CAM as an acid. Because of this, they also differ in other characteristics.

In this lab, the floating disks technique was used in order to measure the efficiency of the photosynthesis. Disks from the leaves of the different plants were cut out using a hole puncher and placed in the syringes. By expanding the syringe, the oxygen and carbon dioxide were sucked out from the leaves. The leaves where after exposed to the light of the different colour. The light color was controlled by placing the colored filter on the light source. When leaves produced enough oxygen to maintain positive buoyancy, they would begin to float. This technique allows to accurately compare the efficiencies of photosynthesis of different plants because, or of the same plants under different colors of light because the changes inside the actual plant were observed, rather than its environment.

Differences in different type of photosynthesis

Name C3 C4 CAM

Compound used 3-carbon chain 4-carbon chain acid

Stomata Open during the day Open during the day Open during the night (and usually closed

during the day)1

Enzyme used for uptake

RUBISCO PEP Carboxylase -

Location of photosynthesis

Throughout the leaf In inner cells -

Adaptive value More efficient under cool and moist

conditions and under normal light

Photosynthesizes faster under high light

intensity and high temperatures.

Has better Water Use Efficiency

Better Water Use Efficiency than C3 plants under arid

conditions.Can survive drought

by CAM-idle

Examples Most of the plants, for example, spinach.

(pic. 2) Spinach

fourwing saltbush, corn

(pic. 3) Fourwing Saltbush

cactuses, agaves

(pic 4) American Agave - typical example of a CAM

plant

1 “The CO2 is converted to an acid and stored during the night. During the day, the acid is broken down and the CO2 is released to RUBISCO for photosynthesis” - http://wc.pima.edu/~bfiero/tucsonecology/plants/plants_photosynthesis.htm

Variables:Independent Variables:

Variable Units and Uncertainty Notes How is it controlled

Type of plant’s photosynthesis

- Three types of plants were used for this experiment, in order to compare the rate of photosynthesis: C3. C4 and CAM

Color of Light Hz; +/- The Color of Light was alternated by placing a filter of different color on the light source

Dependent Variable:

Variable Units and Uncertainty Notes How is it controlled

Time needed for 5 out of 10 leaf disks to float

s +/-s Indirect measurement of the rate of photosynthesis.

Controlled Variable:

Variable Units and Uncertainty Notes How is it controlled

Concentration of sodium Bicarbonate

0.476 mol/dm3 Concentration of sodium bicarbonate in a solution may affect varies the amount of CO2 available for plant to uptake, changing the concentration gradient. Higher concentration in one trial will increase the speed of photosynthesis compared to other trials

Number of disks per trial

# of disks Placing more than 10 leaf disks will decrease the time it will take for 5 of them to float.Placing less than 10 disks will decrease the time it will take for 5 of the them to float.

Leaf disks were counted and ensured to be 10 before being placed into the syringe.

Volume of solution per trial

50 ml +/- 1 ml

Volume of solution per syringe

4ml +/- 0.1 ml A very large amount of water in the syringe would not allow to extract enough oxygen for the from the leaf in order for it to sink because not enough pressure would be produced.

All of the syringes were filled to the same amount before exerting the air from the leaves.

Distance from light 30 cm +/- 0.1 cm Distance from the The distance between

source light source could directly affect the rate of photosynthesis, as light intensity is one of the limiting factors of the photosynthesis. If the light source was closer to the disks, the photosynthesis would appear to happen faster.

the light source and the beakers was measured by a ruler and set up to be 30cm (between the beaker and the actual source not the stand).

Diameter of the disk 6mm +/-0.5 mm Diameter directly affects the surface area of the disks. Large surface area of the disk would increase the make the rate of photosynthesis appear to occur faster than it actually does, because more chloroplasts would be open for photosynthesis.

All of the disks were cut out using the same hole punch.

pH of water - pH is one of the limiting factor of the photosynthesis, because the light-independent reactions require several key enzymes, such as RuBisCO, which denature under a very low or very high pH.

Only distilled water was used for the experiment in order to ensure that its original pH would be 7 at room temperature. Adding sodium bicarbonate will slightly increase the pH of the solution, however, because the same concentration of NaHCO3 was the same in all solutions the pH was constant.

Uncontrolled Variables:

Variable Units and Uncertainty Notes How is it controlled

Mass of the disk mg Mass could affect the experiment in a number of ways: first, it affects the number of chloroplasts available for photosynthesis.

Light Intensity lux Different Light intensities could affect the rate of photosynthesis because light is a limiting factor in photosynthesis. Light intensity could be changed by placing a filter even if the intensity of a light source is constant.

Materials:

Material Quantity Notes

Distilled Water 10l

Sodium Bicarbonate 150g

Low-heat white light source designed for hydroponic plant growth

1

Filters for the light source 5 LEE Filters of the following colours were used for the experiment:

● Red (182)● Yellow (101)● Green (139)● Blue (132)● Violet (049)

Stopwatch (+/- 0.01 s) 1

100 ml beaker (+/- 1 ml) 1

Plants of each type of photosynthesis

5Leaves of each plants were chosen to be big enough to contain surface area enough for at least 5 holes made with hole punch.

C3: Spinach (Spinacia oleracea )C4: Fourwing Saltbush (Atriplex canescens )

CAM: American aloe (Agave americana)

10ml Measuring cylinder (+/- 0.2 ml)

1

5ml Syringe (+/- 0.1 ml) 1

Petri Dish 3 Used to sort the leaf disks.

Electronic Balance (+/- 0.1 ml)

1 Used to measure the mass of sodium bicarbonate

Cardboard box (20cm x 10сm x 10 cm (or bigger))

1 Needs to be big enough to fully cover a syringe

Vernier© Lab Quest 1 Used together with Vernier© Light Intensity Meter to measure the Light intensity of different light colours.

Vernier© Light Intensity Meter

1 Used together with Vernier© Lab Quest to measure the Light intensity of different light colours.

Procedure:1. Preparing the leaf disks:

a. Leaves were collected from all 3 plants. The preference was given to the thinnest what is this word? leaves of about the same thickness in order to diminish the effect of mass on the results of the experiments.

b. Hole Punch was used in order to make 25 leaf disk from every plant.c. Disks made from Spinach were placed in the petri dish.d. Petri dish with spinach disks was labeled C3.e. Disks made from Saltbush were placed in another petri dish.f. Petri dish with spinach disks was labeled C4.g. Disks made from Agave were placed in the last petri dish.h. Petri dish with spinach disks was labeled CAM.

2. Preparing sodium bicarbonate solution a. 50 ml of water were measured using a measuring cylinder and poured into a

beaker.b. 2g of sodium bicarbonate were measured using the electronic balance and then

added to the beaker.c. Syringe was used to gather 4 ml of solution.d. 10 leaf disks were placed inside the syringee. The top of the syringe was closed with fingerf. The pump was pulled several times until the leaf disks sinked.g. Syringe was placed into the box.

3. Setting up the experiment: a. A spot was marked on the table with Xb. A vernier light meter was set up in the spot X.c. The regular light inside the room was turned off.d. Light source was covered with yellow filter and placed 20 cm away from the light

meter.e. The reading on the light meter was read.

4. Conducting an experiment: a. Remove the Light meter from the spot X and replace it with a beaker.b. Syringe was taken out of the box and its contents were placed into the beaker.c. The stopwatch was started.d. The stopwatch was stopped the moment the 5th leaf disk has raised to the surface.

i. If 5 disks have not reached the surface by 10 min, the experiment was stopped and the number of leaf disks that did raise was recorded.

5. Repeating the experiment: a. The experiment was repeated 4 additional times using the yellow filter. (New

carbonate solution was prepared every single time and new leaf disks were used.)

b. Every time a new filter was used, a light meter was set in X and the distance from the light source was alternated on one linear axis, until the reading on the light meter was identical to the original and then the experiment was carried our as before.

c. The was then repeated 20 more times, using the other 4 filters (Red, Green, Blue and Violet. (5 repetitions for every single filter)

6. Experiment for C4 and CAM plants: a. The whole experiment (Steps 2-5) was repeated for the Fourwing Saltbush and the

American aloe

Sources

"Bio Facts: Century Plant." Jacksonville Zoo. Jacksonville Zoo & Garden, n.d. Web. 07 Oct.

2014.

Carter, J. Stein. "Photosynthesis." Photosynthesis. N.p., 14 Jan. 2014. Web. 05 Oct. 2014.

"Types of Photosynthesis." Types of Photosynthesis. N.p., n.d. Web. 06 Oct. 2014.

Pictures Sources:

(pic. 2): http://www.gardeningknowhow.com/wp-content/uploads/2009/03/spinach-plant1.jpg

(pic. 3) http://www.nps.gov/colm/naturescience/images/Four-Wing-Saltbush_1.JPG

(pic. 4): http://www.jacksonvillezoo.org/images/uploads/Agave_american.jpg