Lab Activity 5

Equation

The overall equation for photosynthesis:

light

6CO2 + 6H2O -------------------------> C6H12O6 + 6O2

Source of Oxygen Gas

What is the source of the oxygen produced by the above reaction? Examination of the left hand side of the equation shows that oxygen is found in CO2 and that oxygen is also found in H2O. Conceivably, oxygen could come from either of these sources. Evidence that the source could be H2O came from the studies of C.B. van Niel. Van Niel was investigating the activities of photosynthetic bacteria. One particular group of such bacteria, the purple sulfur bacteria, reduces carbon to carbohydrates during photosynthesis but does not release oxygen. The purple sulfur bacteria require hydrogen sulfide for their photosynthetic activity. In the course of photosynthesis, globules of sulfur accumulate inside the bacterial cells. Van Niel found that the following reaction takes place during photosynthesis in these bacteria:

light

CO2 + 2H2S → (CH2O) + H2O + 2S

On the basis of these findings van Niel proposed the following generalized equation for photosynthesis:

light

CO2 + 2H2A → (CH2O) + H2O + 2A

In this equation, H2A represents an oxidizable substance, such as hydrogen sulfide or free hydrogen. In algae and green plants, however, H2A is water. In short, van Niel proposed that water, not carbon dioxide, was the source of the oxygen in photosynthesis.

More convincing evidence that the O2 released in photosynthesis is derived from

H2O was produced by Ruben, Hassid, and Kamen. The investigators labeled the oxygen in water using a heavy isotope of oxygen (18O2). Later the radioactive oxygen was found in the oxygen gas produced by photosynthesis. This confirmed that the source of the oxygen gas produced during photosynthesis was water.

CO2 + H2O18 → C6H12O6 + O18

Occurrence

Photosynthesis occurs in green plants, Protists (Algae and other pigmented Protists), and bacteria (cyanobacteria and other photosynthetic bacteria).

Autotrophic Nutrition

Photosynthesis is an example of autotrophic nutrition, a form of nutrition in which an organism can manufacture organic substances from simple inorganic precursors. This means that plants can produce their own food. Animals have heterotrophic nutrition, they must eat plants or other animals in order to survive.

Phases

Photosynthesis can be divided into two phases:

1) the light-dependent phase

2) the light-independent phase

Summary of the Light-dependent reaction

In the light-dependent reaction, water is split, producing oxygen gas and hydrogen ions that are associated with electrons. Light energy absorbed by Photosystem I and II is used to boost the electrons along a chain of electron transport molecules. The light-dependent reactions generate a concentration gradient of hydrogen ions between the lumen of the thylakoid membrane and the stroma. The energy released by the flow of hydrogen ions down this gradient is used to produce the chemical energy of ATP. The electrons flow to NADP+, producing reducing power in the form of NADPH.

Summary of the light-independent reactions

In the light-independent reaction, CO2 is fixed by combining it with ribulose bisphosphate and converted into various carbohydrates, using the products of the light-dependent reaction. These products include ATP, which is used as a source of energy, and NADPH, which is used as a source of reducing power.

Nature of Light

White light is composed of a band of colors ranging from violet to red known as the spectrum. Visible light is part of a wider band of radiation known as the electromagnetic spectrum. Light has both the properties of waves and the properties of particles. Photons are packets of light energy. Light is the source of energy for photosynthesis.

CHLOROPLASTS

Photosynthesis takes place in organelles known as chloroplasts. Chloroplasts contain chlorophyll and other photosynthetic pigments, which absorb light energy for photosynthesis. Chloroplasts may be considered the most important of cell organelles, for they are the ultimate sources of most of our food supplies and of our fuel. In higher plants, chloroplasts usually appear as slightly elongated, flattened disks 0.5 to 1.0 µm thick and 2 to 6 µm in diameter.

Chloroplast Structure

1) Surrounded by a double-layered membrane.

2) Contains a system of double-layered membranes known as thylakoids, which are the site of the light-dependent reactions of photosynthesis. The thylakoids are organized into stacks known as grana, which resemble stacks of coins.

3) Chlorophyll, as well as carotene and xanthophyll pigments are embedded within the thylakoid membranes.

4) Stroma - the liquid-filled interior of the chloroplast. The stroma is the site of the light-independent reactions of photosynthesis.

5) Like mitochondria, chloroplasts contain DNA and ribosomes.

The occurrence of DNA and ribosomes within the chloroplast has been used as evidence to support the endosymbiotic theory. The endosymbiotic theory explains the evolution of the chloroplast. According to this theory, the chloroplast was once a free-living photosynthetic bacterial cell. This cell was engulfed by a larger eukaryotic cell. Instead of being digested, the chloroplast survived. It replicated along with the host cell. Over a period of time, the photosynthetic bacterium established a symbiotic relationship with the larger cell. It became important to the host cell because of its ability to produce energy by way of photosynthesis. It eventually evolved into the chloroplast as we know it today.

PHOTOSYNTHETIC PIGMENTS

Pigments are colored molecules that absorb light. Photosynthetic pigments absorb light energy, which is converted to a flow of electrons and then to chemical energy.

Chlorophyll a is the primary photosynthetic pigment in green plants. Chlorophyll a is bluish-green in solution. Accessory pigments absorb wavelengths of light that are not absorbed well by chlorophyll a and thereby extend the range of light available for photosynthesis. The accessory pigments then transmit their energy to chlorophyll a. They include chlorophyll b, carotenoids, and xanthophylls. Chlorophyll b is yellowish-green in solution. Carotenoids are red, orange, and yellow accessory pigments. Xanthophylls are yellow pigments.

Absorption of Light by Photosynthetic Pigments

An absorption spectrum is a graph that shows which wavelengths are most strongly absorbed by a pigment. Chlorophyll a absorbs maximally at 430 and 662 nm. Chlorophyll b absorbs maximally at 443 and 642 nm. Carotenoids absorb maximally at wavelengths between 460 and 550 nm. The action spectrum is a graph showing how the rate of photosynthesis varies with different wavelengths of light. The peaks in the rates of photosynthesis correlate with the peaks in absorption of wavelengths of light by the photosynthetic pigments.

Chlorophyll a and accessory pigments are arranged in light gathering complexes, which are embedded within the thylakoid membrane. These complexes are known as photosystems. Plants contain two light-harvesting systems located in the thylakoid membranes. These systems are designated Photosystem I and Photosystem II.

Each photosystem consists of a reaction-center molecule of chlorophyll a, along with associated molecules.

A photosystem consists of two components:

1) An antenna complex of hundreds of pigment molecules that gather photons and feed the captured light energy to the reaction center; and

2) A reaction center, consisting of one or more chlorophyll molecules in a matrix of protein that passes the energy out of the photosystem.

Absorption of Light by Chlorophyll

When a molecule of chlorophyll absorbs light, the energy in the photon of light is captured by the pigment and used to boost an electron to a higher energy orbital. The boosted electron may be captured by an acceptor molecule. The excitation energy of the electron may then be transmitted to other molecules and transformed into chemical energy that is used to power the synthetic reactions of photosynthesis. If an acceptor does not capture the electron, it returns to its original orbital (ground state) and emits light of a longer wavelength than the light it absorbed. This process is called fluorescence.

Electron transport

Electron transport molecules capture the energy of electrons boosted by chlorophyll and transport them along a chain of molecules, known as the electron transport chain which uses their energy to produce ATP and reduce NADP+ to NADPH.

The transfer of electrons from one electron transport molecule to another involves oxidation-reduction reactions. Oxidation is loss of electrons. An oxidized atom is one that is not carrying as many electrons as it could. Reduction is a gain of electrons. When electrons are added to an atom, it becomes reduced.

The molecules which comprise the electron transport chain include cytochromes, plastoquinones, and plastocyanin.

THE MECHANISM OF THE LIGHT-DEPENDENT REACTION

In the light-dependent reaction, water is split. This produces oxygen gas and hydrogen ions that are associated with electrons. The electrons travel along a chain of electron carrier molecules embedded in the thylakoid membranes. They are boosted along their path by two photosystems (Photosystem I and Photosystem II) using light energy, which is captured by light absorbing pigments. The light-dependent reactions generate a concentration gradient of hydrogen ions between the lumen of the thylakoid membrane and the stroma. The energy released by the flow of hydrogen ions down this gradient is used to produce the chemical energy of ATP. The electrons flow to NADP+, producing reducing power in the form of NADPH.

The Light-Dependent Reaction consists of two stages:

1) The Noncyclic Stage

2) The Cyclic Stage

NONCYCLIC ELECTRON TRANSPORT

A photon of light strikes Photosystem I and the energy is funneled to P700. The energy excites an electron, boosting it from a lower energy orbital to a higher energy orbital. The energized electron is accepted by a membrane-bound electron acceptor known as “X” (Fe4S4). The electrons are then transferred to ferredoxin and then to an enzyme, ferredoxin-NADP reductase. This enzyme then reduces NADP+, converting it to NADPH. The loss of the electron from P700 leaves it with a deficiency of one electron and a resulting positive charge. This electron must be replaced. The electrons removed from the P700 molecule are replaced by electrons derived from Photosystem II.

Photosystem II is associated with the splitting of water and the generation of O2. During photosynthesis the antenna pigments absorb a photon and funnel this energy to the P680 in the reaction center. The P680 enters an excited state and passes one electron to a pheophytin molecule. Pheophytin then passes electrons to quinone (Q). The acceptor molecule Q transfers the electron to an electron transport chain which links Photosystem II and Photosystem I. The chain consists, in sequence of plastoquinone, cytochrome b6/f, and plastocyanin. From plastocyanin the electron moves to Photosystem I, restoring the electron that was lost from P700. The electrons lost by the P680 molecule are replaced by electrons extracted from a water molecule. This splitting of water molecules is known as photolysis.

CYCLIC ELECTRON TRANSPORT

Cyclic Electron Transport involves only Photosystem I. The absorption of light energy causes a flow of electrons from P7OO of excited photosystem I through the electron transport chain following a cyclic path which returns them back to the reaction center in Photosystem I from which they originated, without involving photosystem II. This process is called cyclic electron flow.

In cyclic electron flow, electrons flow from P700 of Photosystem I to ferredoxin. However, instead of continuing along the chain of carriers leading to NADP+, the electron travels to the b6/f complex and eventually returns to Photosystem I, where it started. The b6/f complex uses the energy in the electron to pump a hydrogen ion into the thylakoid lumen. This hydrogen ion can be used to generate ATP. ATP generated by cyclic electron flow is called cyclic photophosphorylation. In this process no reduced NADP+ (NADPH + H+) is formed nor is water split to release oxygen.

THE SYNTHESIS OF ATP

The energy which is used to create ATP comes from the flow of hydrogen ions down their concentration gradient from the lumen of the thylakoid membrane to the stroma side through special channels in the membrane. These channels are complex sets of enzymes that can synthesize ATP from ADP and phosphate; the entire complex is called ATP synthetase, or the CF0 -CF1 complex. The process of ATP production in photosynthesis is referred to as chemiosmosis or photophosphorylation.

LIGHT – INDEPENDENT REACTIONS

The light-independent reactions of photosynthesis are a cyclic series of metabolic reactions in the chloroplast stroma, in which the energy (ATP) and reducing power (NADPH) produced in the light reactions are used to synthesize carbohydrate. In these reactions, CO2 is fixed, that is, it is chemically combined with a molecule known as ribulose- 1, 5-bisphosphate. This molecule is metabolized to produce sugars, which are used for energy and various other carbohydrates. Eventually ribulose bisphosphate is regenerated, completing the cycle.

The light-independent reactions of photosynthesis may be represented by the following reaction:

CO2 + NADPH + H+ + ATP ----------------> glucose + NADP+ + ADP + P

This reaction is also known as the Calvin Cycle, or C3 cycle. The reaction is called the Calvin cycle after Dr. Melvin Calvin of the University of California, the discoverer of the cycle. Dr. Calvin received a Nobel prize in 1961 for his discovery. Dr. Calvin used radioactively-labeled carbon dioxide, 14CO2 to trace the steps in the reactions. Dr. Calvin investigated the process by exposing a culture of green algae, Chlorella to the radioactively labeled CO2. As the plant carried out photosynthesis, it incorporated the radioactively-labeled CO2 into its products. Dr. Calvin exposed the algae to 14CO2 for varying periods of time. He then separated the compounds using chromatography and identified the separated compounds using autoradiography. When Calvin exposed the algae to 14CO2 for 60 seconds, he found that many products were labeled with 14C. In order to identify products closer to the beginning of the reaction, he had to use shorter exposure times. When he tried a 7-second exposure time, most of the radioactivity appeared in a 3-carbon compound, 3-phosphoglycerate (3-PGA). He reasoned that in the first step of the reaction, CO2 must combine with a two-carbon compound. Calvin searched for this two-carbon compound but did not find it.

Calvin began searching for another molecule that combined with CO2 in the initial step of photosynthesis. He was able to identify this molecule. It was a five-carbon molecule named ribulose 1, 5-bisphosphate (RuBP). Dr. Calvin reasoned that the CO2 combines with ribulose 1, 5-bisphosphate. This forms a 6-carbon molecule that is unstable and spontaneously breaks down into two molecules of 3-phosphoglycerate.

Dr. Calvin continued to analyze the products formed at longer intervals of time to separate and identify the remaining products of the light-independent reaction.

MECHANISM OF THE CALVIN CYCLE

(C3 PHOTOSYNTHESIS)

In the first step, CO2 is fixed, that is it combines with a 5-carbon molecule known as ribulose-1, 5-bisphosphate (RuBP). The six-carbon molecule that is formed breaks apart immediately into two three-carbon molecules of 3-phosphoglycerate, hence the name C3 cycle. The enzyme that carries out this reaction is RuBP carboxylase, also known as Rubisco.

In the next step, a phosphate group from the breakdown of ATP is added to 3-phosphoglycerate, converting it to 1, 3-diphosphoglycerate, which is then reduced to glyceraldehyde 3-phosphate. In this reaction a phosphate group is removed from 1, 3-diphosphoglycerate.

Some of the glyceraldehyde 3-phosphate is used to reform RuBP. The rest of the glyceraldehyde 3-phosphate is used to synthesize various sugars and other compounds which are needed by the plant, including fats, amino acids, and nucleic acids.

C4 METABOLISM

C4 metabolism - enables plants to cope with hot, dry environments. The evolution of C4 metabolism has enabled plants to avoid photorespiration, a process that reduces the efficiency of C3 photosynthesis under hot, dry, conditions. Photosynthesis becomes inefficient when the concentration of CO2 decreases and the concentration of O2 increases. This condition results from the closing of the stomata when it is hot and dry to conserve moisture. CO2 cannot enter the leaf and so its concentration decreases. The oxygen produced by photosynthesis cannot leave, so that its concentration increases. Under these conditions, the enzyme RuBP carboxylase begins to combine O2 with ribulose bisphosphate instead of the normal CO2. The products of this oxygen reaction are phosphoglycolic acid, a two-carbon compound, and PGA, a three-carbon compound.

PHOTORESPIRATION:

O2 + RuBP -------------------------> phosphoglycolic acid + PGA

The reactions initiated by rubisco’s fixation of oxygen rather than carbon dioxide are collectively called photorespiration because they occur only in light, consume oxygen, and release carbon dioxide.

Photorespiration greatly reduces the efficiency of RuBP carboxylase. In this process, large amounts of carbon dioxide originally fixed by the enzyme are lost.

Clearly, plants that could avoid photorespiration would have a significant advantage. That advantage was provided by the evolution of C4 photosynthesis.

MECHANISM OF C4 PHOTOSYNTHESIS

In C4 metabolism, the first detectable product of the light-independent reactions is a four-carbon (oxaloacetate) rather than a three-carbon compound (3-phosphoglycerate) as it is in ordinary photosynthesis. C4 plants counteract the effects of photorespiration by compartmentalizing RuBP carboxylase in a site where carbon dioxide concentration is high and oxygen concentration is low.

In C4 plants the stomata are open during the cooler parts of the day, in the early morning and in the evening. They are closed during midday, the hot part of the day.

The reactions of C4 metabolism are a circular set of reactions that take place sequentially between two cells, the bundle sheath cells that surround the vascular bundles and the surrounding mesophyll cells.

Plants with C4 metabolism have a special leaf anatomy known as Krantz Anatomy. The leaf veins are closely spaced. There are no more than four or five cells between adjacent vascular bundles. In these plants, thick-walled bundle sheath cells surround the vascular bundle. These cells are surrounded, in turn, by thin-walled mesophyll cells. The mesophyll is uniform, that is, not distributed as palisade parenchyma and spongy mesophyll. The leaves of plants with Krantz anatomy have two kinds of chloroplasts. In the bundle sheath cells surrounding the veins, there are large chloroplasts, often with few to no grana, and numerous starch grains. The chloroplasts of the mesophyll cells are much smaller, have well-developed grana and usually lack starch grains. Plants with C4 metabolism also have a special enzyme known as PEP Carboxylase. This enzyme, which is found in the mesophyll cells, functions more efficiently under conditions of low CO2 and high O2 than RuBP carboxylase does.

Carbon dioxide diffuses into the leaf through the stomata and combines in the mesophyll cell with a three-carbon compound called phosphoenolpyruvic acid (PEP). This reaction is catalyzed by the enzyme PEP carboxylase. The reaction produces oxaloacetic acid (OAA), or oxaloacetate, a four-carbon acid. Because the first product of these reactions is a four-carbon molecule rather than a 3-carbon molecule as it is in ordinary photosynthesis, this pathway is called C4 metabolism.

C4 PHOTOSYNTHESIS

CO2 + PEP ----------------------------------> oxaloacetic acid

PEP carboxylase

The four-carbon acid is quickly converted to malate or aspartate. The malate or aspartate is then transported from the mesophyll cells to the adjacent bundle-sheath cells. In the bundle sheath cells, the CO2 is split off from the malate or aspartate, leaving a three-carbon compound. Thus, malate and aspartate function as short-lived reservoirs of CO2.

The CO2 released in the bundle-sheath cells is fixed by the Calvin Cycle. Meanwhile, the three-carbon compound is shuttled back to the mesophyll cell where it is converted to PEP.

The pumping by C4 plants of CO2 into bundle-sheath cells keeps the internal concentration of CO2 in bundle-sheath cells high. This high concentration of CO2 increases the effectiveness of the enzyme RuBP carboxylase in combining CO2 and RuBP.

Advantage of C4 Photosynthesis

Plants with C4 photosynthesis are adapted to high light intensities, high temperatures, and dry conditions. They flourish in the tropics. CO2 is released from malate into the bundle-sheath cell causing the buildup of a high concentration of CO2 and a low concentration of O2. This creates suitable conditions for the action of ribulose-bisphosphate carboxylase, the enzyme that fixes CO2. In addition, CO2 liberated by photorespiration in the bundle-sheath cells can be refixed by the C4 pathway operating in the mesophyll cell. The CO2 liberated by photorespiration can thus be conserved instead of being wasted by escaping from the leaf. C4 plants can survive at temperatures that would be lethal to C3 plants. Such plants use carbon dioxide more efficiently. As a result, they are able to carry out photosynthesis at rates comparable to C3 plants, but with smaller stomatal openings and correspondingly less water loss.

Examples of C4 plants include corn, sorghum, sugarcane, millet, crabgrass, and Bermuda grass.

CRASSULACEAN ACID METABOLISM (CAM)

Crassulacean Acid metabolism is so named because it was first discovered in those members of the family Crassulaceae that have succulent leaves. These are plants which live in dry, hot, climates. In order to conserve water, the plants close their stomata during the day and open them at night.

At night, CO2 diffuses through the open stomata into the leaf tissues. The metabolism is almost identical to that in C4 plants: CO2 is attached to PEP (PEP is carboxylated) forming oxaloacetate. The oxaloacetate is then reduced to malate or other acids. These acids are not transported from mesophyll cells to bundle-sheath cells, but are stored within the vacuole of the mesophyll cell. In this way CO2 is stored until daytime. During the day, the malate made the previous night diffuses out of the cell vacuoles in which it was stored. The malate is then decarboxylated, that is, CO2 is removed from the molecule. The liberated CO2 then enters the Calvin Cycle in the typical manner and is fixed by joining it to ribulose bisphosphate. By opening their stomata at night, CAM plants are able to conserve water, avoiding the excessive transpiration loss that would occur in the direct light and heat of the day.

Unlike C4 plants, in which the initial fixation of CO2 occurs in a different cell than in the Calvin Cycle, CAM plants perform all of their photosynthesis in the same cell. Therefore, while the reactions of C4 plants are separated spatially (i.e., they occur in different cells), the reactions of the Crassulacea are separated both spatially (i.e., in different parts of the cell) and temporally (i.e. at different times of the day). Plants with CAM photosynthesis typically do not have a well-defined palisade mesophyll in the leaves, and in contrast to the chloroplasts of the bundle sheath cells of C4 plants those of CAM photosynthesis plants resemble the mesophyll cell chloroplasts of C3 plants.

CAM is more widespread than C4 photosynthesis: it occurs in more than twenty families that include monocots, dicots, and primitive plants.

Examples of CAM plants include cacti, orchids, bromeliads, lilies, and euphorbias.

CAM plants include the maternity plant (Kalanchoë daigremontiana), wax plant (Hoya carnosa), pineapple (Ananas comosus), Spanish moss (Tillandsia usneoides), and at least two genera of ferns (Pyrrosia). Most CAM plants are succulents.

THE SIGNIFICANCE OF PHOTOSYNTHESIS

Photosynthesis may be considered the most important chemical reaction which occurs on the surface of the earth. There are many reasons why photosynthesis is important. Among these reasons are the following:

1) Production of Food

2) Production of O2 and Utilization of CO2 in the Atmosphere

3) Production of Energy