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The vast majority of living organisms obtain their fuel molecules, directly orindirectly, from the process of photosynthesis. The process of photosynthesistransforms light energy into chemical energy, and uses that chemical energy toproduce organic molecules, typically glucose, from water and carbon dioxidemolecules.

These organic molecules are the basis for the macromolecules used for structureand metabolism of living organisms as well as the fuel molecules we use in cellrespiration. The process of photosynthesis also produces oxygen gas as a "by-product", the very same molecule that is used in aerobic cell respiration, withoutwhich most organisms on earth would not survive.

To review: Organisms that obtain energy and carbon from their physicalsurroundings are autotrophs. The vast majority of autotrophs arephotoautotrophs that manufacture their organic molecules by the process ofphotosynthesis. (Organisms that obtain their organic fuel molecules pre-formedfrom the environment are heterotrophs. Animals, fungi, many protists and manybacteria are heterotrophs.) Most photosynthetic organisms are plants or protiststhat contain chlorophyll. Many prokaryotes are also photosynthetic. TheCyanobacteria have chlorophyll pigments; some Bacteria, such as the purple sulfurbacteria, have different light-capturing pigments, and slightly differentphotosynthetic mechanisms. They are studied in microbiology.

Not all autotrophs are photosynthetic; a tiny proportion of our organic fuel ismanufactured by chemosynthetic organisms (or chemoautotrophs).Chemosynthetic autotrophs use energy from chemical reactions involving inorganicatoms and molecules, such as S, Fe, H and N, to make organic compounds.Chemosynthesis is restricted to a very few groups of bacteria, mostly theArchaea. However, chemosynthesis sustains some deep sea-bed ecosystems thatsurround sulfur vents. The chemosynthetic bacteria are also discussed inmicrobiology.

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The Process of PhotosynthesisAs stated, photosynthesis involves the transformation of light energy to chemicalenergy. The chemical energy is then used to manufacture carbohydrate molecules,primarily glucose. In eukaryotic organisms, and in the Cyanobacteria, the processof photosynthesis also produces oxygen. The photosynthetic bacteria produceorganic carbon molecules, but do not produce oxygen. We will discuss the primaryphotosynthetic processes of plants only.

Photosynthesis occurs in all parts of plants that contain the green pigment,chlorophyll, which is located in the chloroplasts. In most plants, however,photosynthesis occurs mostly in leaves, where chloroplasts are concentrated. Inthe laboratory, we shall look closely at the leaf structure as it relates to itsfunction on photosynthesis. At this time we shall turn to the pathways ofphotosynthesis.

Photosynthesis involves two stages, occurring in separate locations withinchloroplasts. In the light-dependent reactions of photosynthesis, light energyis transformed into chemical energy in the form of energy transfer molecules in aseries of re-dox reactions that transfer electrons and hydrogen from water to theenergy transfer molecule NADP+. (Recall that a number of metabolic processes,including cell respiration, involve oxidation-reductions with electrons being "carried"along a gradient of electron transfer molecules.) The light-dependent reactionsare known as photophosphorylations, because they involve producing ATP.They take place on the thylakoid membranes of the grana.

In the Calvin cycle (sometimes called the light-independent reactions), the energyfrom the light reactions is used to manufacture carbohydrate molecules, whichform glucose. These reactions occur in the stroma of the chloroplast.

The two "stages" of photosynthesis are linked by the products of the light-dependent reactions. These products are ATP and NADPH.

The overall chemical equation for photosynthesis* is:

Chlorophyll6CO2 + 12H2O + energy C6H12O6 + 6H2O + 6O2

Chlorophyll(Carbon dioxide + water + light energy glucose + water + oxygen)

* The process of photosynthesis directly produces 12 molecules of the 3-carboncompound, glyceraldehyde-3-phosphate (G3P). Two of these molecules aremetabolized to glucose during a "complete" photosynthesis. The remaining 10molecules of G3P are recycled in the photosynthetic process.

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Photosynthetic RequirementsIn order to do photosynthesis, water, CO2, chlorophyll and light energy must beavailable. We shall briefly discuss each of these requirements.

1 . W a t e rWater is the hydrogen and electron donor for the process ofphotosynthesis. Light energy is used to split water molecules, forming 2H+,2e-, and Oxygen during the process of photosynthesis.

Water is obtained from the environment, absorbed by roots and conductedthroughout the plant in the xylem of the vascular system. Water neededfor photosynthesis is but one of the demands for water in plants. (Howwater moves within plants is discussed in Biology 203.)

2 . Carbon DioxideCarbon dioxide provides the carbon source for manufacturing thecarbohydrates in photosynthesis.

Carbon dioxide diffuses from the atmosphere, through pores in leafsurfaces, called stomata, which are formed by a pair of guard cells. Thecarbon dioxide then diffuses to the photosynthetic cells of the leafmesophyll. The rate of diffusion of carbon dioxide and availability of carbondioxide often limit the rate and amount of photosynthesis that occurs in aplant. (The mechanisms of stomatal operation are discussed in Biology 203,although we will observe stomata in the leaf surfaces in the laboratory.)

3 . ChloroplastsThe pigments needed for photosynthesis are located in the chloroplasts.Recall that the chloroplast has a double membrane with a series of internalstacked membranes. Light energy is captured by the pigments located onthe special membranes in the chloroplast called thylakoids, which arefolded into disk-shaped stacks called grana. The interior compartments ofthylakoids serve as reservoirs for hydrogen ions (H+) that are needed forproducing ATP.

The reactions of photosynthesis that are involved in the transformation oflight energy to chemical energy, the light-dependent reactions, occur onthe thylakoid membranes of the chloroplast.

The reactions needed to produce carbohydrates occur in the stroma regionof the chloroplast. Enzymes are located here. These reactions are known asthe Calvin cycle.

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4 . Light WavesLight is a form of electromagnetic radiation. Visible light is a combination ofmany wavelengths that we can see as different colors (of the rainbow) in therange of 380 - 750 nm. Each wavelength is associated with a specificphoton, or particle of energy. In general, shorter wavelengths have moreenergy.

The light absorbing photosynthetic pigments do not absorb all wavelengths oflight equally. Some light energy cannot be absorbed (and is reflectedinstead) and some is transmitted, or passed through the chloroplasts.

The absorption of different wavelengths, or absorption spectrum, of lightby the photosynthetic pigments can be demonstrated in the laboratory usingspectrophotometers. The light waves most absorbed and most useful tophotosynthesis are reds and blues. Not surprisingly, green light is absorbedpoorly. One can also measure rates of photosynthesis in differentwavelengths to generate an action spectrum. This is done by growingplants in light boxes that have just one wavelength of light.

Absorption Spectrum Action Spectrum

Englemann's Action Spectrum of Spirogyra and Bacteria attracted to the Oxygen evolved.

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5 . The Light Absorbing Pigments of PhotosynthesisChlorophyll is the primary pigment that absorbs light energy inphotosynthesis. In plants, there are two forms of chlorophyll (a, which has amethyl group, and b, which has an aldehyde group) as well as importantaccessory pigments, the carotenes. Each pigment absorbs certainwavelengths, and collects and concentrates light energy for thephotosynthetic process. In addition the carotenes may function to protectchlorophyll molecules from being damaged by too intense light. The red andblue phycocyanin pigments can also absorb light and serve as accessorypigments in photosynthesis.

When pigment molecules absorb energy from light, electrons in the moleculesare excited and moved to a higher energy orbital in an excited state. Theenergy level of a photon absorbed and the rise in energy level to the higherenergy orbital must match, which is why only certain wavelengths can beabsorbed by certain pigments. This rise in energy is temporary. Electronsfall back to their ground state if the energy is not transferred elsewhere.When the electrons fall back to their ground state energy is given off asheat, or sometimes as light (fluorescence). Chlorophyll a does both,fluorescing as red light.

Excitation of Isolated Chlorophyll Molecules

Excitation of Chlorophyll in Chloroplasts

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The photosynthetic pigment molecules do not work alone. They are arrangedon the thylakoids of the chloroplast in clusters of about 300 pigmentmolecules in a protein matrix to form a light-harvesting or antennacomplex that gathers and transfers energy to a photochemical reactioncenter, embedded in a transmembrane protein complex. The reactioncenter has a special chlorophyll a molecule along with the primaryelectron acceptor (which accepts the electrons released from chlorophylla). There are two such complexes found in the chloroplasts, calledPhotosystem I and Photosystem II. Each has a unique electron acceptor.

The reaction centers of Photosystems I and II are activated by slightlydifferent wavelengths of light. The reaction center in Photosystem Iabsorbs light of 700nm best. The reaction center of Photosystem II absorbswavelengths of 680nm. Each thylakoid has thousands of the two differentphotosystems. Both are needed for photosynthesis.

6 . Electron Transport System Molecules (Energy Transfer Molecules)As discussed previously, many chemical reactions of metabolism are coupledoxidation-reductions that utilize a chain of electron transport molecules.These electron carriers specialize in oxidizing and reducing at specific energylevels to minimize energy loss in energy transfers. Both photosynthesis andcellular respiration rely on such molecules. In the process of photosynthesis,the electron transport carriers are embedded in the thylakoid membranes.

Some of the molecules which specialize in these energy transfers in thethylakoids are: NADP+, plastoquinone, two cytochromes, plastocyanin andferredoxin

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The Photosynthesis PathwaysStage I Light (Dependent) Reactions - Cyclic and Non-Cyclic

Photophosphorylation (Electron Flow)• The light-dependent reactions transform light energy into chemical energy that

is trapped and carried by ATP and NADPH to the Calvin Cycle.• The light-dependent reactions require chlorophyll and occur in the thylakoid

membranes of the grana of the chloroplast.• During the light-dependent reactions water is split in an enzyme-mediated

reaction located in photosystem II into:H2O -----> 2H+ + 2e- + 1/2O2

Splitting water produces oxygen and provides electrons (transferred tophotosystem II) and Hydrogen for the reduction of NADP+ to NADPH (NADP gainsH+ and electrons; the water is oxidized because it loses the H+ and e-).

There are two events in the light reactions: noncyclic and cyclic electron flow (ornoncyclic and cyclic photophosphorylation)

1 . Non-Cyclic Photophosphorylation (Non-cyclic Electron Flow)UsesPhotosystem IPhotosystem IIElectron Transport SystemInputs

WaterLight energyEnergy Transfer Molecules

ADP and Pi

NADP+

OutputsATPNADPH (reduced form) (from NADP+, the oxidized form)O2

Organization of the Light Reaction Components in the Thylakoid

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We often diagram the processes of the light reactions to show the relative energylevels of the electrons as they are moved, even though, as shown, the molecules ofphotosystems I and II and the electron transfer molecules are embedded within thethylakoid membranes, organized in a way that best facilitates the process. It isuseful to be familiar with both perspectives.

Light-Dependent Reactions of Photosynthesis or Non-Cyclic Electron Flow

How non-cyclic electron flow works:• Light energy hitting photosystem II excites an electron in the p680 reaction

center causing the chlorophyll molecule to lose an electron to the primaryelectron acceptor. A total of two electrons are actually lost from chlorophyll.The electrons are transferred to the electron transport molecules and passedslowly “down” the chain releasing energy. Some of this energy is used toproduce a H+ gradient within the thylakoid. This gradient ultimately drives themechanism that produces ATP by chemiosmosis.

• The oxidized chlorophyll a molecule needs to replace its electrons. Theelectrons lost by photosystem II are replaced by the splitting of water.Water’s electrons are passed to photosystem II; its H+ is used in the H+ gradient(and can be passed to NADP+) and its oxygen is released as oxygen gasmolecules.

• Light energy also hits photosystem I, exciting its reaction center chlorophyll aelectrons. They are picked up by the primary electron acceptor, transferred toferredoxin and then to NADP+, which will also pick up a H+ forming NADPH.

• The electrons released from photosystem II, once they have passed through theelectron transport system, are used to replace the electrons lost by chlorophyllin photosystem I.

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To summarize, the low energy electrons from water are elevated inenergy by passing through both photosystem II and photosystem I andtrapped by NADP+ where their potential energy will be used for thehigh-energy reduction of carbon in the Calvin cycle. Along the way,ATP, needed for the endergonic Calvin cycle, is also produced.

Non-cyclic electron flow is sometimes referred to as the "Z" scheme, because theelectrons do an energy "zig-zag" as they move from water to photosystem II tophotosystem I to NADP+. The process of using both photosystems has anenhancement effect so that the overall rate and efficiency of light capture ishigher than if there were just one photosystem of if the two photosystems workedindependently of each other.

2 . Cyclic Photophosphorylation (Cyclic Electron Flow)Uses

Photosystem IElectron Transport System

InputsLight energy

OutputsATP (from ADP and Pi)

Cyclic Photophosphorylation only uses Photosystem I and produces only ATP.Cyclic electron flow dates to the earliest photosynthetic bacteria functionedto generate energy, not to facilitate the synthesis of organic fuel molecules.The electrons captured could not be used in the reduction of carbon. Inplants, cyclic photophosphorylation still functions to generate ATP as acomplement to the non-cyclic flow.

In cyclic photophosphorylation, electrons released from the chlorophyll p700are returned back to Photosystem I after passing through the chain ofelectron carriers. ATP is produced by chemiosmosis.

Cyclic Photophosphorylation

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Chemiosmotic synthesis of ATP in the chloroplastIn photosynthesis, the molecules of electron transport system are located in thethylakoid membrane. The energy released from electron transport systems isused to move Hydrogen ions (H+) from the stroma into the inner thylakoidcompartments by active transport. This concentration of H+ in the innercompartment of the thylakoid establishes a concentration and an electricalgradient (the proton-motive force) in the thylakoid compartment that has aninherent (potential) energy value. This is not insignificant. The pH gradient goesfrom pH 5 within the thylakoid membrane to pH 8 in the stroma (a 1000 Xdifference).

The accumulated H+ ions diffuse through the ATP synthase protein complexchannels in the thylakoid membranes that are coupled to ATP synthesis. As the H+

ions flow down the gradient in the protein channels, their energy is used to makeATP from ADP and P on the other side of the thylakoid membrane in the stroma.

Note: Peter Mitchell won a Nobel prize in 1978 for determining the chemiosmotictheory of ATP synthesis. ATP is synthesized in the mitochondria duringcell respiration by a similar mechanism.

Chemiosmotic synthesis of ATP in the Thylakoids

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Stage II The Calvin Cycle and Carbon FixationThe second set of reactions for photosynthesis is known as the Calvin cycle. Thereactions of the Calvin cycle do not use light energy for the energy source. Theyuse the ATP produced in the light reactions for their energy source, and theenergy transfer molecule, NADPH to provide Hydrogen and electrons for thereduction of carbon.

Carbohydrate molecules are produced in Calvin Cycle in the stroma of thechloroplast.

In the Calvin cycle carbon dioxide combines with a 5-carbon sugar (Ribulosebisphosphate) and undergoes a reduction to form 3-carbon molecules. These 3-carbon intermediates can be used to regenerate the 5-carbon sugar or bemetabolized to form the carbohydrate, glucose. They can also be used for thesynthesis of the organic molecules needed for cell structure and function.

The requirements for the Calvin cycle are:• Carbon dioxide (CO2)• NADPH from the light-dependent reactions• ATP from the light-dependent reactions• Ribulose bisphosphate, regenerated in the cycle• Appropriate enzymes for each step in the cycle. Of these, Ribulose

bisphosphate carboxylase/oxidase (Rubisco) is especially important.

The Metabolic Intermediate in Process is:• G3P (Glyceraldehyde-3-Phosphate)

The Calvin cycle produces:• Glucose (carbohydrate)• Ribulose bisphosphate, regenerated in the cycle• Water

The parts of the Calvin cycle are• CO2 Fixation• Reduction• Regeneration• Output or Surplus

The process we are discussing was determined using radioisotopes of 14C. Theresearchers (Calvin, Benson and others) won a Nobel prize for this. This pathwayis called 3-carbon photosynthesis, (because of the 3-C G3P intermediate) todistinguish it from an alternative pathway known as 4-carbon (C-4)photosynthesis, to be discussed in a bit. To some, the Calvin cycle looks like acarbon cut-and-paste dance. It is. It's easy to follow the maze if one countscarbons. It also helps to remember that without this happening, you'd starve!

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Overview of the Calvin Cycle

The "Parts"Carbon Fixation and the Reduction Phase of the Calvin CycleThis must repeat 6 times to form 1 glucose.

CO2 Fixation6CO2 + 6RuBP (Ribulose 1,5-Bisphosphate) 6Hexose Phosphate (not shown) 12 PGA (3-Phosphoglycerate)

Reduction of PGA to G3P12 PGA (3-Phosphoglycerate) +12ATP 12 1,3-Biphosphoglycerate +12NADPH 12 G3P (Glyceraldehyde-3-Phosphate) + (12ADP +12Pi +12NADP+)

Summary (CO2 Fixation and Reduction of PGA to G3P)6CO2 + 6 RuBP + 12 ATP + 12 NADPH 12 G3P (or PGAl).

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Fate of G3PThe 12 molecules of G3P produced will be used for two purposes: Regeneration ofribulose bisphosphate (RuBP) and synthesis of glucose (the output from the cycle).

The Output (Surplus Phase) - Producing GlucoseThe remaining 2 molecules of G3P are converted into one Glucose molecule

2

2 G3P --------------------------------------------------------------------------> 1 Glucose

Although glucose is the typical end product of photosynthesis, plants do not storeor transport glucose. Sucrose (synthesized from fructose-phosphate and glucose-phosphate) is the typical solute translocated throughout the plant, and, as learned,plants typically store starch. In addition, plants are capable of synthesizing all oftheir organic molecules (amino acids, lipids, etc.) from photosyntheticintermediates, notably G3P and glucose phosphate. Plants are very versatile intheir synthetic abilities compared to animals. There are also whole groups of plantproducts, called secondary metabolites, synthesized directly or perhaps as by-products of plant activity. Some secondary metabolites are protective in nature,such as toxins; some, such as lignin, are used in plant structure.

Regeneration of ribulose bisphosphate (RuBP)This will use 10 of the 12 G3P molecules from the reduction phase. Only thecarbon backbone is illustrated. Water, ADP and P are given off in the process.

1 0 + 6 ATP 6 + 6ADP + 6 H2O

Summary10 G3P + 6 ATP ----> 6 RuBP + 6 H2O (+ 6 ADP)

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How the regeneration of RuBP works:(Take G3P in groups of 5 to produce 3 Rubs. Repeat to get 6RuBPs regenerated)

C CC CC C + ATP ----> RuBP

CC

CC CC C remove "head"

CC

C C C CC C C CC C C + ATP ----> RuBP

C C C remove "head" C

C CC CC

CC

C C + ATP ----> RuBPC CC C

C CC CC C + ATP ----> RuBP

CC

CC CC C remove "head"

CC

C C C CC C C CC C C + ATP ----> RuBP

C C C remove "head" C

C CC CC

CC

C C + ATP ----> RuBPC CC C

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Calvin Cycle - the Biochemistry Version:

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Photosynthesis ProductivityIt takes 18 ATP and 12 NADPH to make one molecule of glucose. Much of the lightenergy that hits the surface of plants is not absorbed and not available. And muchof the light that hits earth does not hit photosynthetic surfaces of plants.

The common limit to photosynthesis is the availability of carbon dioxide, whichdiffuses from the atmosphere into the leaves through pores in the leaf surface,called stomata. Unfortunately, these pores also permit diffusion of water fromthe interior of the leaf, and can be a significant source of water loss to the plant.(As much as 90% of the water taken in to a plant is lost this way. Thisevaporation of water through the stomata (called transpiration) is also used bythe plant to generate a tension that serves to pull water up through the xylemfrom the roots to stems and leaves, so this water loss is not a completelynegative thing for the plant. Transpiration will be studied in Biology 203.)

Under hot and dry conditions, many plants must close their stomata to minimizewater loss. During these times the ratio of oxygen to carbon dioxide in the leafincreases, which favors a process that competes with photosynthetic outputcalled photorespiration.

PhotorespirationRubisco, the enzyme that brings CO2 and RuBP together, works only when theconcentration of CO2 is high relative to the level of O2, and when CO2 levels drop,the enzyme, Rubisco, combines RuBP with O2 which is then exported from thechloroplast and broken down into other compounds (in mitochondria andperoxisomes) that actually release CO2 rather than reduce it to PGA -> G3P. TheCalvin cycle is subverted. Photorespiration decreases the photosynthetic output ofthe plant. This reduction in photosynthesis can be significant!

Comparison of CO2 fixation and photorespiration with Rubisco

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C-4 PhotosynthesisSome plants have evolved mechanisms to minimize the effects ofphotorespiration. Such plants have an internal anatomy (the Kranz anatomy) thatseparates the oxygen producing light-dependent reactions of photosynthesis fromthe Calvin cycle. These two processes occur in modified chloroplasts that arelocated in different cells within the leaves. In addition, these plants havealternative methods of trapping and accumulating carbon dioxide so thatphotosynthesis can proceed when stomata are closed. The photosyntheticmechanism used in these plants if called C-4 photosynthesis. The typicalphotosynthesis is called C-3 Photosynthesis.

Modified (Kranz) anatomy of the C-4 plant leafLight reactions occur in leaf mesophyll cells of C-4 plants, just as in C-3 plants.The chloroplasts of the C-4 plant mesophyll cells have lots of grana. Recall thatoxygen is produced only in the light reactions. (C-3 and C-4 leaf structure will bereviewed in lab.)

The Calvin Cycle, however, occurs in the chloroplasts of special bundle sheathcells, cells that surround the veins of the leaf. These chloroplasts have almost nograna, so very little, if any, oxygen is produced in these cells. The bundle sheath isthe outer layer of the leaf veins, which normally add strength to the leaf.

C-3 Leaf Structure C-4 Leaf Structure

Modified carbon dioxide fixation system in the C-4 plant.CO2 combines with RuBP in the first step of the Calvin cycle. In C-4 plants, CO2 isfixed before the Calvin cycle starts. Any CO2 that enters the leaf, at any time,can be combined with a common metabolic intermediate, PEP (phosphoenolpyruvate) in mesophyll cells, forming a 4-carbon acid, Oxaloacetic acid(oxaloacetate) that is converted to malic acid (malate).

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Malate is transferred to the bundle sheath cells where CO2 can be released for re-fixation in the Calvin cycle forming the 3-carbon pyruvate by-product. Pyruvate isreturned to the mesophyll cells where it is converted back to PEP.

This is an efficient trap for CO2 since the 4-carbon acid can accumulate duringnon-light periods, concentrating CO2 when photosynthesis cannot occur, and can beused during periods of low moisture when stomata are closed to prevent waterloss. The separation of Calvin cycle from O2 production raises the efficiency whenO2 levels are high (bright sunlight and higher temperatures). However,regenerating PEP requires ATP, so C-4 photosynthesis may not always be moreproductive than the C-3 pathway, as shown when temperatures are moderate andmoisture levels are adequate. The pathway is genetic; plants can't choose.

C-3 and C-4 Photosynthesis rate comparisons for CO2 and O2 levels and for Temperature

Note C-3 plants perform better at lower temperatures.

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CAM – A Water Conservation MechanismSome C-3 plants close stomata during the daylight hours to conserve water. Theyopen their stomata during the night and use the CO2 acid trap of C-4 plants. Theystore the acids accumulated during the dark in the vacuole. In the daytime, thisCO2 is released for "normal" C-3 photosynthesis, while the stomata can remainclosed to prevent excessive water loss. The name, CAM, is derived from the typesof plants in which it was first discovered, Crassuleans, and for the fact the CO2

trapped forms acids. A significant difference between C-4 plants and CAM plantsis that CAM plants do not have Kranz anatomy. They perform the Calvin cycle inthe same cells as the light reactions. It is strictly a water conservation benefitfor photosynthesis.

Comparison of C-4 and CAM Plants