The production of ATP using the energy of sunlight is called photophosphorylation. Only two sources of energy are available to living organisms: sunlight and oxidation-reduction (redox) reactions. All organisms produce ATP, which is the universal energy currency of life.

In photophosphorylation, light energy is used to create a high-energy electron donor and a lower-energy electron acceptor. Electrons then move spontaneously from donor to acceptor through an electron transport chain.

Contents

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1 Background 2 Cyclic photophosphorylation 3 Noncyclic photophosphorylation

o 3.1 References

[edit] Background

ATP is made by an enzyme called ATP synthase. Both the structure of this enzyme and its underlying gene are remarkably similar in all known forms of life.

ATP synthase is powered by a transmembrane electrochemical potential gradient, usually in the form of a proton gradient. The function of the electron transport chain is to produce this gradient. In all living organisms, a series of redox reactions is used to produce a transmembrane electrochemical potential gradient, or a so-called proton motive force (pmf).

Redox reactions are chemical reactions in which electrons are transferred from a donor molecule to an acceptor molecule. The underlying force driving these reactions is the Gibbs free energy of the reactants and products. The Gibbs free energy is the energy available (“free”) to do work. Any reaction that decreases the overall Gibbs free energy of a system will proceed spontaneously (given that the system is isobaric and also adiabatic)

The transfer of electrons from a high-energy molecule (the donor) to a lower-energy molecule (the acceptor) can be spatially separated into a series of intermediate redox reactions. This is an electron transport chain.

The fact that a reaction is thermodynamically possible does not mean that it will actually occur. A mixture of hydrogen gas and oxygen gas does not spontaneously ignite. It is necessary either to supply an activation energy or to lower the intrinsic activation energy of the system, in order to make most biochemical reactions proceed at a useful rate. Living systems use complex macromolecular structures to lower the activation energies of biochemical reactions.

It is possible to couple a thermodynamically favorable reaction (a transition from a high-energy state to a lower-energy state) to a thermodynamically unfavorable reaction (such as a separation of charges, or the creation of an osmotic gradient), in such a way that the overall free energy of the system decreases (making it thermodynamically possible), while useful work is done at the same time. Biological macromolecules that catalyze a thermodynamically favorable reaction if and only if a thermodynamically unfavorable reaction occurs simultaneously underlie all known forms of life.

Electron transport chains (most known as ETC) produce energy in the form of a transmembrane electrochemical potential gradient. This energy is used to do useful work. The gradient can be used to transport molecules across membranes. It can be used to do mechanical work, such as rotating bacterial flagella. It can be used to produce ATP and NADPH, high-energy molecules that are necessary for growth.

[edit] Cyclic photophosphorylation

In cyclic electron flow, the electron begins in a pigment complex called photosystem I, passes from the primary acceptor to plastoquinone, then to cytochrome b6f (a similar complex to that found in mitochondria), and then to plastocyanin before returning to chlorophyll. This transport chain produces a proton-motive force, pumping H+ ions across the membrane; this produces a concentration gradient that can be used to power ATP synthase during chemiosmosis. This pathway is known as cyclic photophosphorylation, and it produces neither O2 nor NADPH. Unlike non-cyclic photophosphorylation, NADP+ does not accept the electrons, but they are sent back to photosystem I. NADPH is NOT produced in cyclic photophosphorylation. In bacterial photosynthesis, a single photosystem is used, and therefore is involved in cyclic photophosphorylation. It is favoured in anaerobic conditions and conditions of high irradiance and CO2 compensation point.

[edit] Noncyclic photophosphorylation

The other pathway, noncyclic photophosphorylation, is a two-stage process involving two different chlorophyll photosystems. Being a light reaction, Noncyclic photophosphorylation occurs on thylakoid membranes inside chloroplasts. First, a water molecule is broken down into 2H+ + 1/2 O2 + 2e- by a process called photolysis (or light-splitting). The two electrons from the water molecule are kept in photosystem II, while the 2H+ and 1/2O2 are left out for further use. Then a photon is absorbed by chlorophyll pigments on surrounding the reaction core center of the photosystem. The light excites the electrons of each pigment, causing a chain reaction that eventually transfers energy to the core of photosystem II, exciting the two electrons that are transferred to the primary electron acceptor, pheophytin. The deficit of electrons is replenished by taking electrons from another molecule of water. The electrons transfer from pheophytin to plastoquinone, then to plastocyanin, providing the energy for hydrogen ions (H+) to be pumped into the thylakoid space. This creates a gradient, making H+ ions flow back into the stroma of the chloroplast, providing the energy for the regeneration of ATP.

The photosystem II complex replaced its lost electrons from an external source; however, the two other electrons are not returned to photosystem II as they would in the analogous cyclic pathway. Instead, the still-excited electrons are transferred to a photosystem I complex, which boosts their energy level to a higher level using a second solar photon. The highly excited electrons are transferred to the acceptor molecule, but this time are passed on to an enzyme called Ferredoxin- NADP reductase|NADP+ reductase, for short FNR, which uses them to catalyse the reaction (as shown):

NADP+ + 2H+ + 2e- → NADPH + H+

This consumes the H+ ions produced by the splitting of water, leading to a net production of 1/2O2, ATP, and NADPH+H+ with the consumption of solar photons and water.

The concentration of NADPH in the chloroplast may help regulate which pathway electrons take through the light reactions. When the chloroplast runs low on ATP for the Calvin cycle, NADPH will accumulate and the

plant may shift from noncyclic to cyclic electron flow.

Photosynthesis: The Role of LightThe heart of photosynthesis as it occurs in most autotrophs consists of two key processes:

the removal of hydrogen (H) atoms from water molecules the reduction of carbon dioxide (CO2) by these hydrogen atoms to form organic molecules.

The second process involves a cyclic series of reactions named (after its discoverer) the Calvin Cycle. It is discussed in Photosynthesis: Pathway of Carbon Fixation. The details of the first process is our topic here.

A description of some of the experiments that led our understanding of these processes are described in Discovering the Secrets of Photosynthesis.

The electrons (e−) and protons (H+) that make up hydrogen atoms are stripped away separately from water molecules.

2H2O -> 4e− + 4H+ + O2

The electrons serve two functions:

They reduce NADP+ to NADPH for use in the Calvin Cycle. They set up an electrochemical charge that provides the energy for pumping protons from the

stroma of the chloroplast into the interior of the thylakoid.

The protons also serve two functions:

They participate in the reduction of NADP+ to NADPH. As they flow back out from the interior of the thylakoid (by facilitated diffusion), passing down

their concentration gradient), the energy they give up is harnessed to the conversion of ADP to ATP.

Because it is drive by light, this process is called photophosphorylation.

ADP + Pi -> ATP

The ATP provides the second essential ingredient for running the Calvin Cycle.

The removal of electrons from water molecules and their transfer to NADP+ requires energy. The electrons are moving from a redox potential of about +0.82 volt in water to −0.32 volt in NADPH. Thus enough energy must be available to move them against a total potential of 1.14 volts. Where does the needed energy come from? The answer: Light.

The Thylakoid Membrane

Chloroplasts contain a system of thylakoid membranes surrounded by a fluid stroma.

Link to page on chloroplast structure.

Six different complexes of integral membrane proteins are embedded in the thylakoid membrane. The exact structure of these complexes differs from group to group (e.g., plant vs. alga) and even within a group (e.g., illuminated in air or underwater). But, in general, one finds:

1. Photosystem I

The structure of photosystem I in a cyanobacterium ("blue-green alga") has been completely worked out. It probably closely resembles that of plants as well.

It is a homotrimer with each subunit in the trimer containing:

12 different protein molecules bound to 96 molecules of chlorophyll a

o 2 molecules of the reaction center chlorophyll P700 o 4 accessory molecules closely associated with them o 90 molecules that serve as antenna pigments

22 carotenoid molecules 4 lipid molecules 3 clusters of Fe4S4 2 phylloquinones

2. Photosystem IIPhotosystem II is also a complex of

> 20 different protein molecules bound to 50 or more chlorophyll a molecules

o 2 molecules of the reaction center chlorophyll P680 o 2 accessory molecules close to them o 2 molecules of pheophytin (chlorophyll without the Mg++) o the remaining molecules of chlorophyll a serve as antenna pigments.

some half dozen carotenoid molecules. These also serve as antenna pigments. 2 molecules of plastoquinone

3. & 4. Light-Harvesting Complexes (LHC)

LHC-I associated with photosystem I LHC-II associated with photosystem II

These LHCs also act as antenna pigments harvesting light and passing its energy on to their respective photosystems.

The LHC-II of spinach is a homotrimer, with each monomer containing

a single polypeptide 8 molecules of chlorophyll a 6 molecules of chlorophyll b 4 carotenoid molecules

5. Cytochromes b6 and f

6. ATP synthase

How the System Works

Light is absorbed by the antenna pigments of

photosystems II and I. The absorbed energy is transferred to the reaction center chlorophylls, P680 in photosystem II,

P700 in photosystem I. Absorption of 1 photon of light by Photosystem II removes 1 electron from P680. With its resulting positive charge, P680 is sufficiently electronegative that it can remove 1 electron

from a molecule of water. When these steps have occurred 4 times, requiring 2 molecules of water, 1 molecule of oxygen

and 4 protons (H+) are released The electrons are transferred (by way of plastoquinone — PQ in the figure) to the cytochrome

b6/f complex where they provide the energy for chemiosmosis. Activation of P700 in photosystem I enables it to pick up electrons from the cytochrome b6/f

complex (by way of plastocyanin — PC in the figure) and raise them to a sufficiently high redox potential that, after passing through ferredoxin (Fd in the figure),

they can reduce NADP+ to NADPH.

The sawtooth shifts in redox potential as electrons pass from P680 to NADP+ have caused this system to be called the Z-Scheme (although as I have drawn the diagram, it looks more like an "N"). It is also called noncyclic photophosphorylation because it produces ATP in a one-way

process (unlike cyclic photophosphorylation and pseudocyclic photophosphorylation described below).

chemiosmosis in Chloroplasts

chemiosmosis and is an example of facilitated diffusion.

Photophosphorylation

Each CO2 taken up by the Calvin cycle) requires:

o 2 NADPH molecules and o 3 ATP molecules

Each molecule of oxygen released by the light reactions supplies the 4 electrons needed to make 2 NADPH molecules.

The chemiosmosis driven by these 4 electrons as they pass through the cytochrome b6/f complex liberates only enough energy to pump 12 protons into the interior of the thylakoid.

But in order to make 3 molecules of ATP, the ATPase in chloroplasts appears to have 14 protons (H+) pass through it.

So there appears to be a deficit of 2 protons. How is this deficit to be made up? One likely answer: cyclic photophosphorylation.

In cyclic photophosphorylation,

the electrons expelled by the energy of light absorbed by photosystem I pass, as normal, to ferredoxin (Fd).

But instead of going on to make NADPH, they pass to plastoquinone (PQ) and on back into the cytochrome b6/f complex. Here the energy each electron liberates pumps 2 protons (H+) into the interior of the thylakoid

— enough to make up the deficit left by noncyclic photophosphorylation.

This process is truly cyclic because no outside source of electrons is required. Like the photocell in a light meter, photosystem I is simply using light to create a flow of current. The only difference is that instead of using the current to move the needle on a light meter, the chloroplast uses the current to help synthesize ATP.

Pseudocyclic Photophosphorylation

Another way to make up the deficit is by a process called pseudocyclic photophosphorylation in which some of the electrons passing to ferredoxin then reduce molecular oxygen back to H2O instead of reducing NADP+ to NADPH.

At first glance, this might seem a fruitless undoing of all the hard work of photosynthesis. But look again. Although the electrons cycle from water to ferredoxin and back again, part of their pathway is through the chemiosmosis-generating stem of cytochrome b6/f.

Here, then, is another way that simply by turning on a light, enough energy is imparted to electrons that they can bring about the synthesis of ATP.

Antenna Pigments

Chlorophylls a and b differ slightly in the wavelengths of light that they absorb best (although both absorb red and blue much better than yellow and green). Carotenoids help fill in the gap by strongly absorbing green light. The entire complex ensures that most of the energy of light will be trapped and passed on to the reaction center chlorophylls. Photosynthesis is the process by which plants, some bacteria, and some protistans use the energy from sunlight to produce sugar, which cellular respiration converts into ATP, the "fuel" used by all living things. The conversion of unusable sunlight energy into usable chemical energy, is associated with the actions of the green pigment chlorophyll. Most of the time, the photosynthetic process uses water and releases the oxygen that we absolutely must have to stay alive. Oh yes, we need the food as well!

We can write the overall reaction of this process as:

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

Most of us don't speak chemicalese, so the above chemical equation translates as:

six molecules of water plus six molecules of carbon dioxide produce one molecule of sugar plus six molecules of oxygen

Diagram of a typical plant, showing the inputs and outputs of the photosynthetic process. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

Leaves and Leaf Structure | Back to Top

Plants are the only photosynthetic organisms to have leaves (and not all plants have leaves). A leaf may be viewed as a solar collector crammed full of photosynthetic cells.

The raw materials of photosynthesis, water and carbon dioxide, enter the cells of the leaf, and the products of photosynthesis, sugar and oxygen, leave the leaf.

Cross section of a leaf, showing the anatomical features important to the study of photosynthesis: stoma, guard cell, mesophyll cells, and vein. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

Water enters the root and is transported up to the leaves through specialized plant cells known as xylem (pronounces zigh-lem). Land plants must guard against drying out (desiccation) and so have evolved specialized structures known as stomata to allow gas to enter and leave the leaf. Carbon dioxide cannot pass through the protective waxy layer covering the leaf (cuticle), but it can enter the leaf through an opening (the stoma; plural = stomata; Greek for hole) flanked by two guard cells. Likewise, oxygen produced during photosynthesis can only pass out of the leaf through the opened stomata. Unfortunately for the plant, while these gases are moving between the inside and outside of the leaf, a great deal water is also lost. Cottonwood trees, for example, will lose 100 gallons of water per hour during hot desert days. Carbon dioxide enters single-celled and aquatic autotrophs through no specialized structures.

Pea Leaf Stoma, Vicea sp. (SEM x3,520). This image is copyright Dennis Kunkel at www.DennisKunkel.com, used with permission.

The Nature of Light | Back to Top

White light is separated into the different colors (=wavelengths) of light by passing it through a prism. Wavelength is defined as the distance from peak to peak (or trough to trough). The energy of is inversely porportional to the wavelength: longer wavelengths have less energy than do shorter ones.

Wavelength and other saspects of the wave nature of light. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

The order of colors is determined by the wavelength of light. Visible light is one small part of the electromagnetic spectrum. The longer the wavelength of visible light, the more red the color. Likewise the shorter wavelengths are towards the violet side of the spectrum. Wavelengths longer than red are referred to as infrared, while those shorter than violet are ultraviolet.

The electromagnetic spectrum. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

Light behaves both as a wave and a particle. Wave properties of light include the bending of the wave path when passing from one material (medium) into another (i.e. the prism, rainbows, pencil in a glass-of-water, etc.). The particle properties are demonstrated by the photoelectric effect. Zinc exposed to ultraviolet light becomes positively charged because light energy forces electrons from the zinc. These electrons can create an electrical current. Sodium, potassium and selenium have critical wavelengths in the visible light range. The critical wavelength is the maximum wavelength of light (visible or invisible) that creates a photoelectric effect.

Chlorophyll and Accessory Pigments | Back to Top

A pigment is any substance that absorbs light. The color of the pigment comes from the wavelengths of light reflected (in other words, those not absorbed). Chlorophyll, the green pigment common to all photosynthetic cells, absorbs all wavelengths of visible light except green, which it reflects to be detected by our eyes. Black pigments absorb all of the wavelengths that strike them. White pigments/lighter colors reflect all or almost all of the energy striking them. Pigments have their own characteristic absorption spectra, the absorption pattern of a given pigment.

Absorption and transmission of different wavelengths of light by a hypothetical pigment. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

Chlorophyll is a complex molecule. Several modifications of chlorophyll occur among plants and other photosynthetic organisms. All photosynthetic organisms (plants, certain protistans, prochlorobacteria, and cyanobacteria) have chlorophyll a. Accessory pigments absorb energy that chlorophyll a does not absorb. Accessory pigments include chlorophyll b (also c, d, and e in algae and protistans), xanthophylls, and carotenoids (such as beta-carotene). Chlorophyll a absorbs its energy from the Violet-Blue and Reddish orange-Red wavelengths, and little from the intermediate (Green-Yellow-Orange) wavelengths.

Molecular model of chlorophyll. The above image is from http://www.nyu.edu:80/pages/mathmol/library/photo.

Molecular model of carotene. The above image is from http://www.nyu.edu:80/pages/mathmol/library/photo.

Carotenoids and chlorophyll b absorb some of the energy in the green wavelength. Why not so much in the orange and yellow wavelengths? Both chlorophylls also absorb in the orange-red end of the spectrum (with longer wavelengths and lower energy). The origins of photosynthetic organisms in the sea may account for this. Shorter wavelengths (with more energy) do not penetrate much below 5 meters deep in sea water. The ability to absorb some energy from the longer (hence more penetrating) wavelengths might have been an advantage to early photosynthetic algae that were not able to be in the upper (photic) zone of the sea all the time.

The molecular structure of chlorophylls. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

The action spectrum of photosynthesis is the relative effectiveness of different wavelengths of light at generating electrons. If a pigment absorbs light energy, one of three things will occur. Energy is dissipated as heat. The energy may be emitted immediately as a longer wavelength, a phenomenon known as fluorescence. Energy may trigger a chemical reaction, as in photosynthesis. Chlorophyll only triggers a chemical reaction when it is associated with proteins embedded in a membrane (as in a chloroplast) or the membrane infoldings found in photosynthetic prokaryotes such as cyanobacteria and prochlorobacteria.

Absorption spectrum of several plant pigments (left) and action spectrum of elodea (right), a common aquarium plant used in lab experiments about photosynthesis. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

The structure of the chloroplast and photosynthetic membranes | Back to Top

The thylakoid is the structural unit of photosynthesis. Both photosynthetic prokaryotes and eukaryotes have these flattened sacs/vesicles containing photosynthetic chemicals. Only eukaryotes have chloroplasts with a surrounding membrane.

Thylakoids are stacked like pancakes in stacks known collectively as grana. The areas between grana are referred to as stroma. While the mitochondrion has two membrane systems, the chloroplast has three, forming three compartments.

Structure of a chloroplast. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

Stages of Photosynthesis | Back to Top

Photosynthesis is a two stage process. The first process is the Light Dependent Process (Light Reactions), requires the direct energy of light to make energy carrier molecules that are used in the second process. The Light Independent Process (or Dark Reactions) occurs when the products of the Light Reaction are used to form C-C covalent bonds of carbohydrates. The Dark Reactions can usually occur in the dark, if the energy carriers from the light process are present. Recent evidence suggests that a major enzyme of the Dark Reaction is indirectly stimulated by light, thus the term Dark Reaction is somewhat of a misnomer. The Light Reactions occur in the grana and the Dark Reactions take place in the stroma of the chloroplasts.

Overview of the two steps in the photosynthesis process. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

Light Reactions | Back to Top

In the Light Dependent Processes (Light Reactions) light strikes chlorophyll a in such a way as to excite electrons to a higher energy state. In a series of reactions the energy is converted (along an electron transport process) into ATP and NADPH. Water is split in the process, releasing oxygen as a by-product of the reaction. The ATP and NADPH are used to make C-C bonds in the Light Independent Process (Dark Reactions).

In the Light Independent Process, carbon dioxide from the atmosphere (or water for aquatic/marine organisms) is captured and modified by the addition of Hydrogen to form carbohydrates (general formula of carbohydrates is [CH2O]n). The incorporation of carbon dioxide into organic compounds is known as carbon fixation. The energy for this comes from the first phase of the photosynthetic process. Living systems cannot directly utilize light energy, but can, through a complicated series of reactions, convert it into C-C bond energy that can be released by glycolysis and other metabolic processes.

Photosystems are arrangements of chlorophyll and other pigments packed into thylakoids. Many Prokaryotes have only one photosystem, Photosystem II (so numbered because, while it was most likely the first to evolve, it was the second one discovered). Eukaryotes have Photosystem II plus Photosystem I. Photosystem I uses chlorophyll a, in the form referred to as P700. Photosystem II uses a form of chlorophyll a known as P680. Both "active" forms of chlorophyll a function in photosynthesis due to their association with proteins in the thylakoid membrane.

Action of a photosystem. This image is from the University of Minnesota page at http://genbiol.cbs.umn.edu/Multimedia/examples.html.

Photophosphorylation is the process of converting energy from a light-excited electron into the pyrophosphate bond of an ADP molecule. This occurs when the electrons from water are excited by the light in the presence of P680. The energy transfer is similar to the chemiosmotic electron transport occurring in the mitochondria. Light energy causes the removal of an electron from a molecule of P680 that is part of Photosystem II. The P680 requires an electron, which is taken from a water molecule, breaking the water into H+ ions and O-2 ions. These O-2 ions combine to form the diatomic O2 that is released. The electron is "boosted" to a higher energy state and attached to a primary electron acceptor, which begins a series of redox reactions, passing the electron through a series of electron carriers, eventually attaching it to a molecule in Photosystem I. Light acts on a molecule of P700 in Photosystem I, causing an electron to be "boosted" to a still higher potential. The electron is attached to a different primary electron acceptor (that is a different molecule from the one associated with Photosystem II). The electron is passed again through a series of redox reactions, eventually being attached to NADP+ and H+ to form NADPH, an energy carrier needed in the Light Independent Reaction. The electron from Photosystem II replaces the excited electron in the P700 molecule. There is thus a continuous flow of electrons from water to NADPH. This energy is used in Carbon Fixation. Cyclic Electron Flow occurs in some eukaryotes and primitive photosynthetic bacteria. No NADPH is produced, only ATP. This occurs when cells may require additional ATP, or when there is no NADP+ to reduce to NADPH. In Photosystem II, the pumping to H ions into the thylakoid and the conversion of ADP + P into ATP is driven by electron gradients established in the thylakoid membrane.

Noncyclic photophosphorylation (top) and cyclic photophosphorylation (bottom). These processes are better known as the light reactions. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

The above diagrams present the "old" view of photophosphorylation. We now know where the process occurs in the chloroplast, and can link that to chemiosmotic synthesis of ATP.

Chemiosmosis as it operates in photophosphorylation within a chloroplast. Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

Halobacteria, which grow in extremely salty water, are facultative aerobes, they can grow when oxygen is absent. Purple pigments, known as retinal (a pigment also found in the human eye) act similar to chlorophyll. The complex of retinal and membrane proteins is known as bacteriorhodopsin, which generates electrons which establish a proton gradient that powers an ADP-ATP pump, generating ATP from sunlight without chlorophyll. This supports the theory that chemiosmotic processes are universal in their ability to generate ATP.

Dark Reaction | Back to Top

Carbon-Fixing Reactions are also known as the Dark Reactions (or Light Independent Reactions). Carbon dioxide enters single-celled and aquatic autotrophs through no specialized structures, diffusing into the cells. Land plants must guard against drying out (desiccation) and so have evolved specialized structures known as stomata to allow gas to enter and leave the leaf. The Calvin

Cycle occurs in the stroma of chloroplasts (where would it occur in a prokaryote?). Carbon dioxide is captured by the chemical ribulose biphosphate (RuBP). RuBP is a 5-C chemical. Six molecules of carbon dioxide enter the Calvin Cycle, eventually producing one molecule of glucose. The reactions in this process were worked out by Melvin Calvin (shown below).

The above image is from http://www-itg.lbl.gov/ImgLib/COLLECTIONS/BERKELEY-LAB/PEOPLE/INDIVIDUALS/index/BIOCHEM_523.html, Ernest OrlandoLawrence Berkeley National Laboratory. " One of the new areas, cultivated both in Donner and the Old Radiation Laboratory, was the study of organic compounds labeled with carbon-14. Melvin Calvin took charge of this work at the end of the war in order to provide raw materials for John Lawrence's researches and for his own study of photosynthesis. Using carbon-14, available in plenty from Hanford reactors, and the new techniques of ion exchange, paper chromatography, and radioautography, Calvin and his many associates mapped the complete path of carbon in photosynthesis. The accomplishment brought him the Nobel prize in chemistry in 1961. (The preceding information was excerpted from the text of the Fall 1981 issue of LBL Newsmagazine.) Citation Caption: LBL News, Vol.6, No.3, Fall 1981 Melvin Calvin shown with some of the apparatus he used to study the role of carbon in photosynthesis."

The first steps in the Calvin ccycle. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinaue The first stable product of the Calvin Cycle is phosphoglycerate (PGA), a 3-C chemical. The energy from ATP and NADPH energy carriers generated by the photosystems is used to attach phosphates to (phosphorylate) the PGA. Eventually there are 12

molecules of glyceraldehyde phosphate (also known as phosphoglyceraldehyde or PGAL, a 3-C), two of which are removed from the cycle to make a glucose. The remaining PGAL molecules are converted by ATP energy to reform 6 RuBP molecules, and thus start the cycle again. Remember the complexity of life, each reaction in this process, as in Kreb's Cycle, is catalyzed by a

different reaction-specific enzyme.

The rate of photosynthesis is affected by a number of factors including light levels, temperature, availability of water, and availability of nutrients. If the conditions that the plant needs are improved the rate of photosynthesis should increase.

The maximum rate of photosynthesis will be constrained by a limiting factor. This factor will prevent the rate of photosynthesis from rising above a certain level even if other conditions needed for photosynthesis are improved. This limiting factor will control the maximum possible rate of the photosynthetic reaction.

For instance, increasing the temperature from 10ºC to 20ºC could double the rate of photosynthesis as the plant's enzymes will be closer to their optimum working temperature. As the temperature is increased, molecules in the cells will be moving at a faster rate due to kinetic theory. If the temperature is raised above a certain level, the rate of photosynthesis will drop as the plant's enzymes are denatured. They will therefore be more likely to join onto the enzymes and react.

The amount of water available to the plant will affect the rate of photosynthesis. If the plant does not have enough water, the plant's stomata will shut and the plant will be deprived of CO². It is difficult in normal lab conditions to prove that water directly affects photosynthesis unless a heavy isotope is used to trace the path of water.

Chlorophyll is needed for photosynthesis. This can be proved by studying a variegated leaf. It is however very difficult to study how different levels of chlorophyll in the plant will affect it's photosynthesis rate. This is because in a variegated leaf the cells either contain chlorophyll or they don't.

Carbon dioxide concentration will directly affect the rate of photosynthesis as it is used in the photosynthesis reaction. It is also easy to change the amount of carbon dioxide that the plant receives.

Light is also directly used in the photosynthesis reaction and is easy to change in normal lab conditions. Carbon Dioxide and Light are the factors that I will change in the experiment as they are easy to change and measure.

Apparatus Needed For The Experiments

1. Elodea 2. 20mm² syringe 3. Capillary tubing 4. Stand 5. Stopwatch

6. Ruler 7. NaHCO³ Solution 8. Bench lamp 9. Distilled water

Method

I could measure the decrease in the substances needed for photosynthesis, such as how much the amount of CO2 decreases over time. This is however difficult in normal lab conditions. I will instead measure how one of the products of photosynthesis (oxygen) increases over time. I am planning to use the following method for my experiment.

1. The apparatus is set up as below with the syringe full of the 0.01M solution of NaHCO3 solution. Two marks 10cm apart are made on the capillary tubing.

2. The syringe is placed 0.05m away from the lamp. 3. Using the syringe plunger the meniscus of the NaHCO3 is set so that it is level with the first

mark. 4. A stopwatch is then started. The meniscus should gradually move down the capillary tube as the

elodea produces oxygen as a by-product of photosynthesis. As the oxygen is produced it increases the pressure in the syringe and so the meniscus is pushed down the tube.

5. When the meniscus reaches the level of the bottom mark the stopwatch should be stopped and the time should be noted in a table such as the one below.

Molarity of NaHCO3

Light Intensity 1/d² (m)

0.00(Distilled water)

0.01 0.02 0.05 0.07 0.1

400

278

204

156

100

25

11

4

The light intensities have been worked out using the following equation

Light Intensity = 1 / Distance² (m)

6. Using the same piece of elodea and the same distance between the lamp and the syringe the experiment (steps 1 to 5) should be repeated for the other concentration of NaHCO3.7. The experiment (steps 1 to 6) should then be repeated at each different distance between the syringe and the light for all the NaHCO3 concentrations. The remaining distances are 0.05m, 0.06m, 0.07m, 0.08m, 0.1m, 0.2m, 0.3m, and 0.5m.8. The entire experiment should then be repeated three times in order to obtain more accurate data and to get rid of any anomalies that may occur in a single experiment.

Measuring the volume of oxygen is more accurate than counting the number of bubbles produced as each bubble could be a different size. In order to make this experiment as accurate as possible a number of steps must be taken.

The experiment should be carried out in darkness with only the light from the bench lamp reaching the elodea.

The same piece of elodea should be used each time in order to make sure that each experiment is being carried out with the same leaf surface area.

The amount of NaHCO3 solution should be the same for each experiment. 20mm² should be used each time.

The lamp should be at the same height for each experiment. It should be level with the syringe each time.

The distance should be measured from the front of the lamp to the syringe. Although taking these steps will make the experiment more accurate, it's accuracy is still limited by several factors.

Some of the oxygen will be used for photosynthesis by the plant. Some of the oxygen will dissolve into the water.

From these recorded times I will work out the rate of the reaction using the following equation.

Rate Of the Reaction = 1 / Time (s)

Using these rates I plan to plot a graph of the rate of reaction against light intensity.

Predictions

Light

I predict that if the light intensity increases the rate of the reaction will increase at a proportional rate until a certain level is reached, the rate of increases will then go down. Eventually a level will be reached where increasing the light intensity will have no more effect on the rate of reaction as there is some other limiting factor.

Light is needed for photosynthesis in plants. When chloroplasts in the leaf's cell are exposed to light they synthesise ATP from ADP. Oxygen is produced as a by-product of the photosynthesis reaction. Therefore increasing the concentration of light will increase the amount of ATP being synthesised from ADP and so more oxygen will be released as a by product.

NaHCO3

I predict that as the concentration of NaHCO3 increases the rate of the reaction will increase at a proportional rate. Eventually increasing the NaHCO3 concentration more will have no effect as other limiting factors will be limiting the rate of photosynthesis. Carbon dioxide is needed for the photosynthesis reaction. It is used to make the organic products of photosynthesis. If the elodea is able to absorb more CO2 then the rate of photosynthesis will increase as the plant is able to make more of the organic compounds. The plant is given CO2 in the form of NaHCO3.

Results

Pooled results from the group were used. They were taken over a 2 day period.

Molarity of NaHCO3

Light Intensity 1/d² (m)

0.00(Distilled water)

0.01 0.02 0.05 0.07 0.1

400 3571 1666 1099 523 200 243

278 1670 5183 988 600 375 262

204 4998 4485 1175 1005 473 351

156 5590 2300 1770 1445 621 550

100 9990 3150 2900 2552 1224 645

25 4762 3984 2850 1640 1408

11 5945 4348 3780 2830 2564

4 16480 11904 5196 6578 3226

Using these results I worked out the rate

Rate Of the Reaction = 1 / Time(s) x 1000

The rate was multiplied by 1000 to make the numbers easier to handle.

Molarity of NaHCO3

Light Intensity 1/d² (m)

0.00(Distilled water)

0.01 0.02 0.05 0.07 0.1

400 0.28 0.60 0.91 1.91 5.00 4.12

278 0.60 0.19 1.01 1.67 2.67 3.82

204 0.20 0.22 0.85 1.00 2.11 2.85

156 0.18 0.43 0.56 0.69 1.61 1.82

100 0.10 0.32 0.34 0.39 0.82 1.55

25 0.21 0.25 0.35 0.61 0.71

11 0.17 0.23 0.26 0.35 0.39

4 0.06 0.08 0.19 0.15 0.31

A graph of the rate of reaction against light intensity was drawn. It shows how the amount of CO2 and light affect the rate of photosynthesis. Lines of best fit were drawn for each CO2 concentration to make up for any inaccuracy in any individual result. The line of best fit gives a good picture of how the overall rate of reaction is affected by the light and CO2.

Interpretation

I will analyse the results for how the amount of light and CO2 affects the rate of photosynthesis.

My prediction that the rate of photosynthesis would go up if the light intensity and NaHCO3 levels were increased proved correct. As the elodea absorbed the light and CO2 it produced oxygen gas which increased the pressure in the syringe. This pushed the air bubble in the capillary tube down. The chloroplasts produce ATP and reduce NADP to NADPH2 when exposed to light. It is at this stage of the reaction that oxygen is produced as a waste product.

As predicted when the light intensity increases so does the rate of photosynthesis. I predicted that a level would be reached where increasing the light intensity would have no more effect on the rate of reaction as there would be some other limiting factor which limits the rate of the reaction. The rate increases at a steady rate as the light intensity increases until near the end of each line where the rate of increase decreases. This is either because the photosynthesis reaction has reached it's maximum rate of reaction or another factor is limiting the rate. As 6 different CO2 concentrations were used I can see that the first five reactions are not occurring at their maximum rate as there is the 0.1M NaHCO3 rest which is occurring at a faster rate then the other 5. The photosynthesis reactions of the other five test must therefore be limited by the concentration of CO2 to the plant.

As predicted when the NaHCO3 concentration is increased the plant in able to get more CO2 which causes the rate of reaction to go up. I predicted that once the NaHCO3 had been raised above a certain level increasing the rate further would have no effect as there would be other limiting factors limiting the rate of the reaction. As the NaHCO3 concentration in the water was increased the rate of photosynthesis was able to go up. The plant therefore made more oxygen as

a waste product. At a NaHCO3 concentration of 0.1M once the light intensity gets above 300 the rate of reaction slows down very quickly. This could be because photosynthesis is occurring at it's maximum possible rate or because another limiting factor is limiting the rate of reaction.

Distilled Water

With the distilled water the rate of reaction went up from 0.1 to 0.4 when the light intensity was increased from 100 to 400. This is a 4 times rise which is quite large. The curve on the graph does however level out quite soon showing that the rate is being limited by the lack of NaHCO3 in the water.

0.01M NaHCO3

At a light intensity of 4 the rate is 0.06 but this rises to 0.6 when the light intensity is brought up to 400. The curve is very shallow and levels off towards a light intensity of 350 - 400.

0.02M NaHCO3

The amount of NaHCO3 is double that of the 0.01M NaHCO3 experiment. The rate also finishes off twice that of the 0.01M experiment. This would surgest that there was a directly proportional relationship between the amount of NaHCO3 and the rate of reaction.

0.05M NaHCO3

The curve for the 0.05M NaHCO3 is steeper than the previous curves. The rate rises to 1.9 at a light intensity of 400.

0.07M NaHCO3

The 0.07M NaHCO3 test produces a line which is steeper than all the previous curves. The plant is using the extra CO2 to photosynthesise more. As the plant has more CO2 the limiting factor caused by the lack of CO2 is reduced. This test did produce a big anomaly. The rate for a light intensity of 400 is 5. By following the line of best fit I can see that this result should be more like 3.5. The elodea for this test was very close to the light source. It is possible that it had been left here for a while which caused the lamp to heat the elodea up. This would have increased the rate of reaction of the plant's enzymes which would have increased the photosynthesis rate.

0.1M NaHCO3

The 0.1M NaHCO3 produced the steepest line. Near the end of the line it looks as if the rate of reaction is hit by another limiting factor. The line goes up steadily but then between a light

intensity of 300 and 400 levels off very quickly. This would surgest that at a 0.1M NaHCO3 is sufficient for the plant to photosynthesise at it's maximum rate with it's current environmental conditions. Increasing the NaHCO3 concentration after this level would therefore have no effect unless the next limiting factor was removed.

The fact that the curve levels off so quickly indicates that there is another limiting factor limiting the photosynthesis. It could be temperature. These tests are being carried out at room temperature so the temperature would have to be raised another 15ºC before the enzymes in the plant's cells were at their optimum working temperature. More tests could be done by using water that was at a higher temperature to see what effect this would have on the photosynthesis rate. It is however impossible to raise the plant's temperature without affect other factors. For instance the actual amount of oxygen released by the plant is slightly more than the readings would surgest as some of the oxygen would dissolve into the water. At a higher temperature less oxygen would be able to dissolve into the water so the readings for the photosynthesis rate could be artificially increased.

It is also possible that the photosynthetic reactions in the plant are occurring at their maximum possible rate and so can not be increased any more.

The light is probably not a limiting factor as all but one of the curves level off before the maximum light intensity of 400 is reached. The maximum light intensity that the plants can handle is therefore just below 400.

Water will not be a limiting factor as the plants are living in water. They therefore have no stomata and absorb all their CO2 by diffusion through the leaves.

r Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

The first stable product of the Calvin Cycle is phosphoglycerate (PGA), a 3-C chemical. The energy from ATP and NADPH energy carriers generated by the photosystems is used to attach phosphates to (phosphorylate) the PGA. Eventually there are 12 molecules of glyceraldehyde phosphate (also known as phosphoglyceraldehyde or PGAL, a 3-C), two of which are removed from the cycle to make a glucose. The remaining PGAL molecules are converted by ATP energy to reform 6 RuBP molecules, and thus start the cycle again. Remember the complexity of life, each reaction in this process, as in Kreb's Cycle, is catalyzed by a

different reaction-specific enzyme.