BIOL 1010 – FALL 2024

PHOTOSYNTHESIS
LAB # 4 – INTRODUCTION

Objectives:
- Improve knowledge of respiration and photosynthesis.
- Evaluate how light and darkness affect photosynthesis.
- Review the use of a compound light microscope to observe plant
epidermis.

Introduction:
All living organisms require energy for their metabolic (chemical) processes. While most
organisms produce energy via the process of respiration using carbohydrates to
produce ATP, the ultimate source of this energy is the sun. Photosynthetic organisms,
including plants, protists (single-celled organisms), and blue-green algae
(cyanobacteria), convert light energy into the chemical energy of carbohydrates, which
can be used to power metabolism.

Cellular respiration:
Cells require ATP to survive because ATP is the
main source of energy in the cell. Molecules of
ATP are produced through the catabolism of
carbohydrates, proteins and fats. Respiration is
a series of oxidation steps which releases energy
stored in macromolecules (such as glucose) and
converts it into energy-rich molecules (such as
ATP) that can then be used to perform cellular
work.

Cellular respiration starts with glycolysis. During

glycolysis, glucose molecules (C6H12O6) are
broken down into two molecules of pyruvate
(C3H3O3). This reaction takes place in the cytoplasm of the cell and results in the
production of 2 ATP molecules via substrate-level phosphorylation. Additional energy is
stored into two molecules of the coenzyme NADH. Oxygen is not required for this
process.

Pyruvate can then be further degraded via several metabolic pathways. The pathway
used depends on whether an inorganic electron acceptor is present or not.

Under aerobic conditions, molecular oxygen is present as an electron acceptor and

aerobic respiration occurs. Pyruvate is transported into the mitochondria where it is

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converted into acetyl CoA. Acetyl CoA is further oxidized in the citric acid cycle. These
processes result in the complete oxidation of pyruvate into three energy-poor CO2
molecules, the formation of one molecule of ATP, and the storage of most of the
released energy in the form of four NADH and one FADH2 molecules. The recovery of
the energy stored in these coenzymes takes place in the inner mitochondrial membrane
where the electrons are transported, via the electron transport chain, to the final
electron acceptor O2 to form water. This creates a proton gradient that is used for the
synthesis of as many as 38 ATP molecules per glucose molecule oxidized.

The global equation for the aerobic respiration is:

Note: Although this process is very efficient in producing large amounts of ATP
molecules, about 55% of the energy is “lost” in the form of heat. In warm-blooded
animals, the heat released by this process is used to maintain body temperature.

Photosynthesis:
The general equation for photosynthesis is:

Note that photosynthesis produces organic molecules (glucose) from inorganic one
(carbon dioxide).

The conversion of carbon dioxide into carbohydrate

can be divided in a two-step process:
- A light reaction where light energy is harvested
and is converted to chemical energy in the form of
ATP and NADPH.
- A light-independent reaction where the chemical
energy ATP and NADPH are used in the Calvin
cycle to convert carbon dioxide to carbohydrate.

Let’s look at those reactions in more details

Light-Dependent Reaction:
The light-dependent reactions take place within the thylakoid membranes and are
dependent on solar energy from white light (visible light). White light consists of multiple

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wavelengths of light: violet, blue, green, yellow, orange and red. The chlorophyll
pigments within the thylakoid membranes can only harvest energy from red and blue
wavelengths of light.

Steps of the Light-Dependent Reactions:

1. When light is absorbed by one of the many pigments in photosystem II (PSII),

energy is passed inward from pigment to pigment until it reaches the reaction center.
There, energy is transferred to P680, boosting an electron to a high-energy level.
The high-energy electron is passed to an acceptor molecule that forward it to the
electron transport chain.
2. The electron that was removed from PSII is replaced by an electron obtained from
the splitting of water. The splitting of water releases 2 electrons so two molecules of
water need to be split to release a molecule of oxygen.

10% of the oxygen produced is used by the plant, the rest is released in the
atmosphere where it is used by aerobic organisms (such as us!) to support
respiration. The process also releases 4 protons (H+) in the thylakoid interior.

3. In the electron transport chain, the high-energy electron from PSII is transferred first
to a small organic molecule (plastoquinone, Pq), then to a cytochrome complex
(Cyt), and finally to a copper-containing protein called plastocyanin (Pc). As the

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 electron moves through this electron transport chain, it goes from a higher to a lower
energy level, releasing energy. Some of the energy is used to pump protons (H+)
from the stroma (outside of the thylakoid) into the thylakoid interior.

4. When the excited electron from photosystem II reaches the end of the electron
transport chain it is now a low-energy electron. It will be excited again by solar
energy when passing through photosystem I (PSI).

5. This high-energy electron from PSI is passed to a protein called ferredoxin (Fd) and
then to NADP reductase where the electron along with a proton is added to NADP+
to create NADPH (reduction).

6. By now, the transfer of H+ from the electron transport chain along with the release of
H+ from the splitting of water, has formed a proton gradient where the [H+] in the
thylakoid interior is greater than the [H+] in the stroma. The energy built in the pH
gradient is used to make ATP.

7. The only route available for the H+ ions to move back into the stroma and release
the concentration gradient is through the ATP synthase enzyme. The enzyme
harnesses the energy associated with the flow of protons to make ATP from ADP
and phosphate (Pi). This process of making ATP using energy stored in a chemical
gradient is called chemiosmosis.

8. Both the ATP and NADPH created by the light-dependent reactions will be used by
the light-independent reactions which converts CO2 into carbohydrates.

Light-Independent Reactions:
The light-independent reactions take place within the stroma of the chloroplasts.
Steps of the Light-Independent Reactions (Calvin cycle):

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1. During the first step of the process the Rubisco enzyme adds CO2 to ribulose-1,5-
bisphosphate (RuBP) creating a six-carbon compound that is split into two three-
carbon molecules 3-phospho-glyceric acid (3PG).

2. The second step is a reduction reaction where 3PG is reduced to glyceraldhyde-3-

phosphate (G3P). Energy from ATP and electrons from NADPH are used in this
process to form six G3P molecules from 3 RuBP and 3 CO2 molecules. Out of these
six molecules only one will be diverted for the synthesis of a glucose molecule. The
other five G3P are used in the regeneration step (step 3) of 3 RuBP molecules so
that a new cycle can begin. Note that step 3 will also requires ATP.

3. So, three carbons enter the cycle as CO2 and three carbons exit the cycle as G3P.

Also, for every 2 G3P that are used to make glucose (over two Calvin cycles), 10
G3P are used to produce more RuBP so that the light-independent reactions can
continue.

4. During the process, ATP is converted back to ADP + Pi and NADPH is oxidized back
to NADP+. These products are recycled by the light-dependent reactions in order to
make new ATP and NADPH molecules.

Where does photosynthesis takes place?

Chloroplasts are organelles found in photosynthetic eukaryotes such as plants and

algae. These organelles are where photosynthesis takes place. Like mitochondria,
chloroplasts are believed to be of bacterial origin as they possess their own DNA. They
are also surrounded by two membranes; the outer membrane which is permeable to
small organic molecules, while the inner membrane is less permeable but possesses
transport proteins that allow for the transport of selected molecules. The innermost
matrix of chloroplasts, called the stroma, contains metabolic enzymes and multiple
copies of the chloroplast genome as well as starch grains. When excess glucose is

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produced, the molecules form chains in a specific pattern, known as starch. Starch is
therefore the storage form of glucose.

Chloroplasts also have a third internal membrane called the thylakoid membrane, which
is extensively folded and appears as stacks of flattened disks when observed using
electron micrographs. The thylakoids contain the light-harvesting complex, including
pigments such as chlorophyll, as well as the electron transport chains used in
photosynthesis.

Leaf structure and Location of chloroplasts in the leaf:

The figure below shows a drawing of the cross section of a leaf; the function of the
difference cells is listed below:

 Cuticle - the waxy, water-repelling layer on the top and bottom surfaces of a leaf; it
helps keep the leaf from dying out and protects it from invading bacteria, insects,
and fungi. The cuticle is secreted by the epidermis.
 Upper epidermis - the protective, outer layer of cells on the upper surface of a leaf,
usually one cell thick. The epidermis secretes the waxy cuticle.

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 Mesophyll - the chlorophyll-containing leaf tissue located between the upper and
lower epidermis. These cells convert sunlight into usable chemical energy for the
plant. It is made of:
o Palisade mesophyll - a layer of elongated cells located under the upper
epidermis. These cells contain most of the leaf's chlorophyll, converting sunlight
into usable chemical energy for the plant.
o Spongy mesophyll - the layer below the palisade mesophyll; it has irregularly-
shaped cells with many air spaces between the cells. These cells contain some
chlorophyll. The spongy mesophyll cells communicate with the guard cells
(stomata), causing them to open or close, depending on the concentration of
gases in the leaf.
o Air space - intercellular gaps within the spongy mesophyll. These gaps are
filled with gas that the plant uses (carbon dioxide - CO2) and gases that the
plant is expelling (oxygen - O2, and water vapor).
 Vein (vascular bundle) - Veins provide support for the leaf and transport both
water and minerals (via xylem) and food energy (via phloem) through the leaf and to
the rest of the plant.
 Lower epidermis - the waxy skin (outermost cells) on the underside of a leaf,
usually one cell thick; it keeps the leaf from drying out.
 Guard cell - one of a pair of sausage-shaped cells that surround a stoma (a pore in
a leaf). Guard cells change shape (as light and humidity change), causing the
stoma to open and close.
 Stoma - (plural stomata) a pore (or opening) in a leaf where water vapor and other
gases leave and enter the plant. Stomata are formed by two guard cells that
regulate the opening and closing of the pore.

Stomata:

Stomata are found on leaves, stems and

petals of flowers and are an important
plant structure affecting photosynthesis.
They consist of specialized cells, called
guard cells that surround a tiny pore
called a stoma. Their main function is to
allow gases such as carbon dioxide, water
vapor and oxygen to move rapidly into
and out of the leaf. Stomata are therefore
very important for photosynthesis as they
control the amount of carbon dioxide that
can enter the cells and therefore the rate

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of the light-independent reaction. So, you are probably thinking that it would be
advantageous for cells to keep their stomata open at all time.

However, stomata are also the structures where water can exit the plant due to
transpiration. Because water is the main media required for all metabolic activity in the
plant, losing too much water by transpiration
is synonymous with death of the plant. The
plant is then faced with the dilemma of
leaving their stomata open enough to allow
carbon dioxide to get in the leaf tissue for
photosynthesis while keeping them close to
prevent water loss by transpiration. One way
plants are dealing with this issue is by
minimizing the size of the opening during
the hot period of the day. Another way will
be evident when you look at leave samples
under the microscope during your
laboratory…

Using a microscope to measure cellular size using the ocular micrometer

(a review)
The size of the specimen viewed under a microscope can be accurately determined by
using an ocular micrometer. The ocular micrometer is a graduated scale that is
located in one of the eyepieces of your microscope. This scale does not have any units
and has been calibrated for each objective lens using a fixed “ruler” known as the stage
micrometer. The distances between two tick marks of the ocular micrometer (OM unit)
for each objective lens has been calculated and are mentioned in the Table below:

Lens used µm/ocular micrometer unit

4X 25
10X 10
40X 2.5
100X 1

So, if you specimen length is 13 OM units when observed using the 10X objective lens,
the actual size of the specimen will be equal:
13 OM units x 10 µm/OM unit = 130 µm

Magnification and scale bar

When drawing a cell viewed under the microscope, it is important to also indicate the
actual size of the cell. This is commonly done by inserting a scale bar at the bottom
right corner of the drawing, which indicates the relationship between the size of the

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drawing and the actual size of the specimen. To determine the size of the scale bar and
the corresponding actual size, you first need to calculate the magnification of the
drawing. The magnification of the drawing is calculated using the following formula:

Note: The size of the drawing and the actual size of the specimen must have the
same units (i.e. both should be in cm, mm or µm).

Note: The magnification of the microscope (ocular x objective) is

different from the magnification of a drawing (ratio of size of drawing
to actual size of specimen). Do not mix them up!

The scale-bar can then be calculated by adapting the formula used to calculate the
magnification, as followed:

- You can decide that your scale-bar is going to be 0.8 cm in length,

Actual size corresponding to 0.8 cm in the drawing (in cm) =

0.8cm / Magnification

You would then draw a scale-bar (a line at the bottom right corner of your
drawing) that is 0.8 cm in length, and write the value from the above calculation
on top of the bar (making sure to include the units).

- If you want to calculate the scale-bar that corresponds to 100 µm in actual size (for
example), the formula is modified as follows:

Scale- bar corresponding to 100 µm actual size (in µm) = Magnification x

100µm

In this case, you would draw a scale-bar the length of the value you just
calculated and write 100 µm on top of the bar.

Examples of microscopy pictures with scale-bars placed at the right corner of the image

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 PROTOCOL

This week, you will perform two experiments to investigate the production of
oxygen gas by photosynthesis on spinach leaves and the consumption of CO2 by
Elodea plants in an aquatic environment.

You will also have a chance to practice microscopic techniques again while
viewing leaf epidermis, where you will be able to observe the stomata and guard
cells, which are responsible for allowing gas exchange in and out of the leaf.

Part I: Investigate oxygen production by photosynthetic spinach disk

Small disks of spinach, from which the air has been removed, will be sank into two
beakers with buffer. One beaker will be place in the dark; one will be placed
under light. Record what happens and explain the results in relation to what you
know from the photosynthetic process.

Procedure:

1. A solution of 0.2% NaHCO3 has been prepared for you. This solution will act as
a pH buffered environment to keep your leaf disks healthy throughout the
experiment.
Explain in your lab assignment how to prepare the solution (must be done
before the lab).
2. Using a hole puncher, cut out 30 disks from the provided spinach leaves.
These should be cut over a 50 mL beaker half filled with the buffer solution.
3. Transfer the disks and buffer into a 10 mL syringe. Add buffer until the
syringe is about 3/4 full.
4. Insert the plunger, point the tip of the syringe upwards and push all of the air
out, being sure not to douse everyone in your vicinity.
5. Now, put your index finger over the syringe tip and pull back on the plunger to
create a vacuum within the syringe. Try pulling back 1-2 cm to start with; be
gentle, you do not want to damage the leaf disks. You should see bubbles
forming around the edges of the leaf disks. Make sure the leaf disks remain in
the buffer.

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6. While still under vacuum, remove your finger from the syringe tip. As the
vacuum is released, the leaf disks will refill with buffer.
7. Tap the syringe several times to see if the leaf disks sink or float.
8. Repeat steps 4-7 until all or most of your disks sink. You may need to adjust
the amount of vacuum you are creating if your disks do not sink after 3-4
trials. Your goal is to get the disks to the point where they JUST sink. Note that
applying too much vacuum could result in damaging the disks.
9. Pour the disks into a Petri dish containing some buffer.
10. Transfer the disks that sink into two 50 mL beakers half-filled with buffer.
Use the edge of a pair of forceps to slide under and grab the disks and sink
them. Be careful not to crush them. Put 10 disks into each beaker.
11. Place one beaker under a light source and one beaker in a dark drawer.
12. After 10 mins, record how many of your disks float in the two beakers in
Table 1.
13. Continue the incubation for a total of 30 min, record the # of discs floating
after 20 min and 30 min incubation in Table 1.
14. Explain the process observed. How does this relate to photosynthesis?

Part II: Investigate the effect of light and darkness in Elodea leaves
Below is a basic protocol for an Elodea investigation that will allow you to
indirectly observe the use of carbon dioxide by the plant when exposed to light or
darkness. Because this experiment requires a 24h incubation period, a picture of
the tubes will be provided to you. Please read the protocol carefully so you can
understand and analyze the results properly. Pre-lab questions highlighted in red
must be completed before attending the lab.

Part A: Done for you

1. Prepare a solution of phenol red by adding 6-8 drops of concentrated phenol
red to 70 mL of water (NOTE: Do NOT use buffer for this experiment).

Phenol red is a pH indicator that will be orange at neutral pH; yellow in acidic
environment and dark red in basic environment.

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 The amount of phenol red added will depend on the acidity/basicity of the tap
water used.

2. If your solution is not orange, use a straw to gently blow air into the solution
until the solution reaches a neutral pH.

3. Transfer 15 ml of solution into four test tubes, labeled No Elodea-Light (L), No

Elodea-Dark (D) and Elodea-light (EL) or Elodea-Dark (ED).

4. Place a cut 3 cm piece of Elodea stem (including leaves) into EL and ED tubes.

5. Cover your test tubes with Parafilm to minimize reactions with the ambient
air.

6. Place your test tubes in the appropriate treatment areas and leave them
under those conditions for 24 h.

7. Record the color of the tubes as shown on the picture provided during the
presentation in Table 2.

Part B:
To understand and analyze this experiment, you will need to investigate the color
change of the pH indicator (phenol red) when an acid or a base is added to the
solution. So, the following experiment is performed:

1. Prepare a solution of phenol red by adding 2-3 drops of concentrated phenol

red to 20 mL of water until the color of the solution is orange. (NOTE: Do NOT
use buffer for this experiment).

The amount of phenol red added will depend on the acidity/basicity of the tap
water used.

2. Transfer 10 ml of solution into two test tubes, labeled Acid (A) and Base (B).

3. Add the acidic solution drop by drop to tube A; vortex after each addition and
observe the change in color. Record the number of drops added and the color
obtained in Table 3.

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4. Add the basic solution drop by drop to tube B; vortex after each addition and
observe the change in color. Record the number of drops added and the color
obtained in Table 3.

5. From the results obtained, determined what happened to the Elodea tubes
placed under the light or in darkness (see Table 2) and answer the questions.

Part III: Examine the leaf epidermis to determine the location of the
stomata using a compound light microscope

In this exercise, you will observe the leaf upper and lower epidermis for the
presence of stomata (and chloroplast).
a. To prepare a lower epidermis wet mount, take a leaf so that the lower
epidermis is facing down and the upper epidermis is facing up. Fold the leaf
downwards so that the lower epidermis is toward the inside of the fold; the
upper epidermis should crack. Gently pull one of the folded portions
downwards (you can use tweezers for this). At the torn edge you should see
a transparent tissue; this is the lower epidermis. Cut this portion from the
rest of the leaf, place it in a drop of water on a slide and add a coverslip on
top of the drop.

b. To prepare an upper epidermis wet mount, take a leaf so that the lower
epidermis is facing down and the upper epidermis is facing up. This time,
fold the leaf upwards so that the upper epidermis is inside the fold; the
lower epidermis should crack. Pull one of the folded portions upwards; you
should see a translucent tissue at the torn edge. This is the upper
epidermis. Cut this portion from the rest of the leaf, place it in a drop of
water on a slide and add a coverslip.

c. Examine the two epidermal peels under the microscope and draw your
observations in Figure 1. Make sure you include a title to your figure, label
the structures you can identify, place a scale-bar in the lower right corner of
each drawings and answer the questions.

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 Appendix: Review the use of a compound light microscope

Please review lab # 1 carefully and watch the video on Canvas, as you will be
required to show proficiency in using the microscope before using it in for your
experiment.

Using a Microscope (Summary)

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