8.21: Light-Independent Reactions - Biology

After the energy from the sun is converted into chemical energy and temporarily stored in ATP and NADPH molecules, the cell has the fuel needed to build carbohydrate molecules for long-term energy storage. Where does the carbon come from? It comes from carbon dioxide, the gas that is a waste product of respiration in microbes, fungi, plants, and animals.

In plants, carbon dioxide (CO2) enters the leaves through stomata, where it diffuses over short distances through intercellular spaces until it reaches the mesophyll cells. Once in the mesophyll cells, CO2 diffuses into the stroma of the chloroplast—the site of light-independent reactions of photosynthesis. These reactions actually have several names associated with them. Another term, the Calvin cycle, is named for the man who discovered it, and because these reactions function as a cycle. Others call it the Calvin-Benson cycle to include the name of another scientist involved in its discovery. The most outdated name is dark reactions, because light is not directly required (Figure 1). However, the term dark reaction can be misleading because it implies incorrectly that the reaction only occurs at night or is independent of light, which is why most scientists and instructors no longer use it.

The light-independent reactions of the Calvin cycle can be organized into three basic stages: fixation, reduction, and regeneration.

Stage 1: Fixation

In the stroma, in addition to CO2, two other components are present to initiate the light-independent reactions: an enzyme called ribulose bisphosphate carboxylase (RuBisCO), and three molecules of ribulose bisphosphate (RuBP), as shown in Figure 2. RuBP has five atoms of carbon, flanked by two phosphates.

Practice Question

In stage 1, the enzyme RuBisCO incorporates carbon dioxide into an organic molecule, 3-PGA. In stage 2, the organic molecule is reduced using electrons supplied by NADPH. In stage 3, RuBP, the molecule that starts the cycle, is regenerated so that the cycle can continue. Only one carbon dioxide molecule is incorporated at a time, so the cycle must be completed three times to produce a single three-carbon GA3P molecule, and six times to produce a six-carbon glucose molecule.

Which of the following statements is true?

  1. In photosynthesis, oxygen, carbon dioxide, ATP, and NADPH are reactants. GA3P and water are products.
  2. In photosynthesis, chlorophyll, water, and carbon dioxide are reactants. GA3P and oxygen are products.
  3. In photosynthesis, water, carbon dioxide, ATP, and NADPH are reactants. RuBP and oxygen are products.
  4. In photosynthesis, water and carbon dioxide are reactants. GA3P and oxygen are products.

[reveal-answer q=”542141″]Show Answer[/reveal-answer]
[hidden-answer a=”542141″]Answer d is true.[/hidden-answer]

RuBisCO catalyzes a reaction between CO2 and RuBP. For each CO2 molecule that reacts with one RuBP, two molecules of another compound (3-PGA) form. PGA has three carbons and one phosphate. Each turn of the cycle involves only one RuBP and one carbon dioxide and forms two molecules of 3-PGA. The number of carbon atoms remains the same, as the atoms move to form new bonds during the reactions (3 atoms from 3CO2 + 15 atoms from 3RuBP = 18 atoms in 3 atoms of 3-PGA). This process is called carbon fixation, because CO2 is “fixed” from an inorganic form into organic molecules.

Stage 2: Reduction

ATP and NADPH are used to convert the six molecules of 3-PGA into six molecules of a chemical called glyceraldehyde 3-phosphate (G3P). That is a reduction reaction because it involves the gain of electrons by 3-PGA. Recall that a reduction is the gain of an electron by an atom or molecule. Six molecules of both ATP and NADPH are used. For ATP, energy is released with the loss of the terminal phosphate atom, converting it into ADP; for NADPH, both energy and a hydrogen atom are lost, converting it into NADP+. Both of these molecules return to the nearby light-dependent reactions to be reused and reenergized.

Stage 3: Regeneration

Interestingly, at this point, only one of the G3P molecules leaves the Calvin cycle and is sent to the cytoplasm to contribute to the formation of other compounds needed by the plant. Because the G3P exported from the chloroplast has three carbon atoms, it takes three “turns” of the Calvin cycle to fix enough net carbon to export one G3P. But each turn makes two G3Ps, thus three turns make six G3Ps. One is exported while the remaining five G3P molecules remain in the cycle and are used to regenerate RuBP, which enables the system to prepare for more CO2 to be fixed. Three more molecules of ATP are used in these regeneration reactions.

This link leads to an animation of the Calvin cycle. Click stage 1, stage 2, and then stage 3 to see G3P and ATP regenerate to form RuBP.

Try It

During the evolution of photosynthesis, a major shift occurred from the bacterial type of photosynthesis that involves only one photosystem and is typically anoxygenic (does not generate oxygen) into modern oxygenic (does generate oxygen) photosynthesis, employing two photosystems. This modern oxygenic photosynthesis is used by many organisms—from giant tropical leaves in the rainforest to tiny cyanobacterial cells—and the process and components of this photosynthesis remain largely the same. Photosystems absorb light and use electron transport chains to convert energy into the chemical energy of ATP and NADH. The subsequent light-independent reactions then assemble carbohydrate molecules with this energy.

Photosynthesis in desert plants has evolved adaptations that conserve water. In the harsh dry heat, every drop of water must be used to survive. Because stomata must open to allow for the uptake of CO2, water escapes from the leaf during active photosynthesis. Desert plants have evolved processes to conserve water and deal with harsh conditions. A more efficient use of CO2 allows plants to adapt to living with less water. Some plants such as cacti (Figure 3) can prepare materials for photosynthesis during the night by a temporary carbon fixation/storage process, because opening the stomata at this time conserves water due to cooler temperatures. In addition, cacti have evolved the ability to carry out low levels of photosynthesis without opening stomata at all, a mechanism to face extremely dry periods.

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These reactions are closely coupled to the thylakoid electron transport chain as the energy required to reduce the carbon dioxide is provided by NADPH produced in photosystem I during the light dependent reactions. The process of photorespiration, also known as C2 cycle, is also coupled to the calvin cycle, as it results from an alternative reaction of the RuBisCO enzyme, and its final byproduct is another glyceraldehyde-3-P.

The Calvin cycle, Calvin–Benson–Bassham (CBB) cycle, reductive pentose phosphate cycle (RPP cycle) or C3 cycle is a series of biochemical redox reactions that take place in the stroma of chloroplast in photosynthetic organisms.

Photosynthesis occurs in two stages in a cell. In the first stage, light-dependent reactions capture the energy of light and use it to make the energy-storage and transport molecules ATP and NADPH. The Calvin cycle uses the energy from short-lived electronically excited carriers to convert carbon dioxide and water into organic compounds [4] that can be used by the organism (and by animals that feed on it). This set of reactions is also called carbon fixation. The key enzyme of the cycle is called RuBisCO. In the following biochemical equations, the chemical species (phosphates and carboxylic acids) exist in equilibria among their various ionized states as governed by the pH.

The enzymes in the Calvin cycle are functionally equivalent to most enzymes used in other metabolic pathways such as gluconeogenesis and the pentose phosphate pathway, but they are found in the chloroplast stroma instead of the cell cytosol, separating the reactions. They are activated in the light (which is why the name "dark reaction" is misleading), and also by products of the light-dependent reaction. These regulatory functions prevent the Calvin cycle from being respired to carbon dioxide. Energy (in the form of ATP) would be wasted in carrying out these reactions that have no net productivity.

The sum of reactions in the Calvin cycle is the following:

Hexose (six-carbon) sugars are not a product of the Calvin cycle. Although many texts list a product of photosynthesis as C
6 H
12 O
6 , this is mainly a convenience to counter the equation of respiration, where six-carbon sugars are oxidized in mitochondria. The carbohydrate products of the Calvin cycle are three-carbon sugar phosphate molecules, or "triose phosphates", namely, glyceraldehyde-3-phosphate (G3P).

Steps Edit

In the first stage of the Calvin cycle, a CO
2 molecule is incorporated into one of two three-carbon molecules (glyceraldehyde 3-phosphate or G3P), where it uses up two molecules of ATP and two molecules of NADPH, which had been produced in the light-dependent stage. The three steps involved are:

  1. The enzyme RuBisCO catalyses the carboxylation of ribulose-1,5-bisphosphate, RuBP, a 5-carbon compound, by carbon dioxide (a total of 6 carbons) in a two-step reaction. [5] The product of the first step is enediol-enzyme complex that can capture CO
    2 or O
    2 . Thus, enediol-enzyme complex is the real carboxylase/oxygenase. The CO
    2 that is captured by enediol in second step produces an unstable six-carbon compound called 2-carboxy 3-keto 1,5-biphosphoribotol (CKABP [6] ) (or 3-keto-2-carboxyarabinitol 1,5-bisphosphate) that immediately splits into 2 molecules of 3-phosphoglycerate (also written as 3-phosphoglyceric acid, PGA, 3PGA, or 3-PGA), a 3-carbon compound. [7]
  2. The enzyme phosphoglycerate kinase catalyses the phosphorylation of 3-PGA by ATP (which was produced in the light-dependent stage). 1,3-Bisphosphoglycerate (glycerate-1,3-bisphosphate) and ADP are the products. (However, note that two 3-PGAs are produced for every CO
    2 that enters the cycle, so this step utilizes two ATP per CO
    2 fixed.)
  3. The enzyme glyceraldehyde 3-phosphate dehydrogenase catalyses the reduction of 1,3BPGA by NADPH (which is another product of the light-dependent stage). Glyceraldehyde 3-phosphate (also called G3P, GP, TP, PGAL, GAP) is produced, and the NADPH itself is oxidized and becomes NADP + . Again, two NADPH are utilized per CO
    2 fixed.

The next stage in the Calvin cycle is to regenerate RuBP. Five G3P molecules produce three RuBP molecules, using up three molecules of ATP. Since each CO
2 molecule produces two G3P molecules, three CO
2 molecules produce six G3P molecules, of which five are used to regenerate RuBP, leaving a net gain of one G3P molecule per three CO
2 molecules (as would be expected from the number of carbon atoms involved).

The regeneration stage can be broken down into steps.

    converts all of the G3P reversibly into dihydroxyacetone phosphate (DHAP), also a 3-carbon molecule. and fructose-1,6-bisphosphatase convert a G3P and a DHAP into fructose 6-phosphate (6C). A phosphate ion is lost into solution.
  1. Then fixation of another CO
    2 generates two more G3P.
  2. F6P has two carbons removed by transketolase, giving erythrose-4-phosphate (E4P). The two carbons on transketolase are added to a G3P, giving the ketose xylulose-5-phosphate (Xu5P).
  3. E4P and a DHAP (formed from one of the G3P from the second CO
    2 fixation) are converted into sedoheptulose-1,7-bisphosphate (7C) by aldolase enzyme.
  4. Sedoheptulose-1,7-bisphosphatase (one of only three enzymes of the Calvin cycle that are unique to plants) cleaves sedoheptulose-1,7-bisphosphate into sedoheptulose-7-phosphate, releasing an inorganic phosphate ion into solution.
  5. Fixation of a third CO
    2 generates two more G3P. The ketose S7P has two carbons removed by transketolase, giving ribose-5-phosphate (R5P), and the two carbons remaining on transketolase are transferred to one of the G3P, giving another Xu5P. This leaves one G3P as the product of fixation of 3 CO
    2 , with generation of three pentoses that can be converted to Ru5P.
  6. R5P is converted into ribulose-5-phosphate (Ru5P, RuP) by phosphopentose isomerase. Xu5P is converted into RuP by phosphopentose epimerase.
  7. Finally, phosphoribulokinase (another plant-unique enzyme of the pathway) phosphorylates RuP into RuBP, ribulose-1,5-bisphosphate, completing the Calvin cycle. This requires the input of one ATP.

Thus, of six G3P produced, five are used to make three RuBP (5C) molecules (totaling 15 carbons), with only one G3P available for subsequent conversion to hexose. This requires nine ATP molecules and six NADPH molecules per three CO
2 molecules. The equation of the overall Calvin cycle is shown diagrammatically below.

RuBisCO also reacts competitively with O
2 instead of CO
2 in photorespiration. The rate of photorespiration is higher at high temperatures. Photorespiration turns RuBP into 3-PGA and 2-phosphoglycolate, a 2-carbon molecule that can be converted via glycolate and glyoxalate to glycine. Via the glycine cleavage system and tetrahydrofolate, two glycines are converted into serine + CO
2 . Serine can be converted back to 3-phosphoglycerate. Thus, only 3 of 4 carbons from two phosphoglycolates can be converted back to 3-PGA. It can be seen that photorespiration has very negative consequences for the plant, because, rather than fixing CO
2 , this process leads to loss of CO
2 . C4 carbon fixation evolved to circumvent photorespiration, but can occur only in certain plants native to very warm or tropical climates—corn, for example.

Products Edit

The immediate products of one turn of the Calvin cycle are 2 glyceraldehyde-3-phosphate (G3P) molecules, 3 ADP, and 2 NADP + . (ADP and NADP + are not really "products." They are regenerated and later used again in the Light-dependent reactions). Each G3P molecule is composed of 3 carbons. For the Calvin cycle to continue, RuBP (ribulose 1,5-bisphosphate) must be regenerated. So, 5 out of 6 carbons from the 2 G3P molecules are used for this purpose. Therefore, there is only 1 net carbon produced to play with for each turn. To create 1 surplus G3P requires 3 carbons, and therefore 3 turns of the Calvin cycle. To make one glucose molecule (which can be created from 2 G3P molecules) would require 6 turns of the Calvin cycle. Surplus G3P can also be used to form other carbohydrates such as starch, sucrose, and cellulose, depending on what the plant needs. [8]

These reactions do not occur in the dark or at night. There is a light-dependent regulation of the cycle enzymes, as the third step requires reduced NADP.

There are two regulation systems at work when the cycle must be turned on or off: the thioredoxin/ferredoxin activation system, which activates some of the cycle enzymes and the RuBisCo enzyme activation, active in the Calvin cycle, which involves its own activase.

The thioredoxin/ferredoxin system activates the enzymes glyceraldehyde-3-P dehydrogenase, glyceraldehyde-3-P phosphatase, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, and ribulose-5-phosphatase kinase, which are key points of the process. This happens when light is available, as the ferredoxin protein is reduced in the photosystem I complex of the thylakoid electron chain when electrons are circulating through it. [9] Ferredoxin then binds to and reduces the thioredoxin protein, which activates the cycle enzymes by severing a cystine bond found in all these enzymes. This is a dynamic process as the same bond is formed again by other proteins that deactivate the enzymes. The implications of this process are that the enzymes remain mostly activated by day and are deactivated in the dark when there is no more reduced ferredoxin available.

The enzyme RuBisCo has its own, more complex activation process. It requires that a specific lysine amino acid be carbamylated to activate the enzyme. This lysine binds to RuBP and leads to a non-functional state if left uncarbamylated. A specific activase enzyme, called RuBisCo activase, helps this carbamylation process by removing one proton from the lysine and making the binding of the carbon dioxide molecule possible. Even then the RuBisCo enzyme is not yet functional, as it needs a magnesium ion bound to the lysine to function. This magnesium ion is released from the thylakoid lumen when the inner pH drops due to the active pumping of protons from the electron flow. RuBisCo activase itself is activated by increased concentrations of ATP in the stroma caused by its phosphorylation.

41 Overview of Photosynthesis

By the end of this section, you will be able to do the following:

  • Explain the significance of photosynthesis to other living organisms
  • Describe the main structures involved in photosynthesis
  • Identify the substrates and products of photosynthesis

Photosynthesis is essential to all life on earth both plants and animals depend on it. It is the only biological process that can capture energy that originates from sunlight and converts it into chemical compounds (carbohydrates) that every organism uses to power its metabolism. It is also a source of oxygen necessary for many living organisms. In brief, the energy of sunlight is “captured” to energize electrons, whose energy is then stored in the covalent bonds of sugar molecules. How long lasting and stable are those covalent bonds? The energy extracted today by the burning of coal and petroleum products represents sunlight energy captured and stored by photosynthesis 350 to 200 million years ago during the Carboniferous Period.

Plants, algae, and a group of bacteria called cyanobacteria are the only organisms capable of performing photosynthesis ((Figure)). Because they use light to manufacture their own food, they are called photoautotrophs (literally, “self-feeders using light”). Other organisms, such as animals, fungi, and most other bacteria, are termed heterotrophs (“other feeders”), because they must rely on the sugars produced by photosynthetic organisms for their energy needs. A third very interesting group of bacteria synthesize sugars, not by using sunlight’s energy, but by extracting energy from inorganic chemical compounds. For this reason, they are referred to as chemoautotrophs .

The importance of photosynthesis is not just that it can capture sunlight’s energy. After all, a lizard sunning itself on a cold day can use the sun’s energy to warm up in a process called behavioral thermoregulation. In contrast, photosynthesis is vital because it evolved as a way to store the energy from solar radiation (the “photo-” part) to energy in the carbon-carbon bonds of carbohydrate molecules (the “-synthesis” part). Those carbohydrates are the energy source that heterotrophs use to power the synthesis of ATP via respiration. Therefore, photosynthesis powers 99 percent of Earth’s ecosystems. When a top predator, such as a wolf, preys on a deer ((Figure)), the wolf is at the end of an energy path that went from nuclear reactions on the surface of the sun, to visible light, to photosynthesis, to vegetation, to deer, and finally to the wolf.

Main Structures and Summary of Photosynthesis

Photosynthesis is a multi-step process that requires specific wavelengths of visible sunlight, carbon dioxide (which is low in energy), and water as substrates ((Figure)). After the process is complete, it releases oxygen and produces glyceraldehyde-3-phosphate (GA3P), as well as simple carbohydrate molecules (high in energy) that can then be converted into glucose, sucrose, or any of dozens of other sugar molecules. These sugar molecules contain energy and the energized carbon that all living things need to survive.

The following is the chemical equation for photosynthesis ((Figure)):

Although the equation looks simple, the many steps that take place during photosynthesis are actually quite complex. Before learning the details of how photoautotrophs turn sunlight into food, it is important to become familiar with the structures involved.

Basic Photosynthetic Structures

In plants, photosynthesis generally takes place in leaves, which consist of several layers of cells. The process of photosynthesis occurs in a middle layer called the mesophyll . The gas exchange of carbon dioxide and oxygen occurs through small, regulated openings called stomata (singular: stoma), which also play roles in the regulation of gas exchange and water balance. The stomata are typically located on the underside of the leaf, which helps to minimize water loss due to high temperatures on the upper surface of the leaf. Each stoma is flanked by guard cells that regulate the opening and closing of the stomata by swelling or shrinking in response to osmotic changes.

In all autotrophic eukaryotes, photosynthesis takes place inside an organelle called a chloroplast . For plants, chloroplast-containing cells exist mostly in the mesophyll. Chloroplasts have a double membrane envelope (composed of an outer membrane and an inner membrane), and are ancestrally derived from ancient free-living cyanobacteria. Within the chloroplast are stacked, disc-shaped structures called thylakoids . Embedded in the thylakoid membrane is chlorophyll, a pigment (molecule that absorbs light) responsible for the initial interaction between light and plant material, and numerous proteins that make up the electron transport chain. The thylakoid membrane encloses an internal space called the thylakoid lumen . As shown in (Figure), a stack of thylakoids is called a granum , and the liquid-filled space surrounding the granum is called stroma or “bed” (not to be confused with stoma or “mouth,” an opening on the leaf epidermis).

On a hot, dry day, the guard cells of plants close their stomata to conserve water. What impact will this have on photosynthesis?

The Two Parts of Photosynthesis

Photosynthesis takes place in two sequential stages: the light-dependent reactions and the light-independent reactions. In the light-dependent reactions , energy from sunlight is absorbed by chlorophyll and that energy is converted into stored chemical energy. In the light-independent reactions , the chemical energy harvested during the light-dependent reactions drives the assembly of sugar molecules from carbon dioxide. Therefore, although the light-independent reactions do not use light as a reactant, they require the products of the light-dependent reactions to function. In addition, however, several enzymes of the light-independent reactions are activated by light. The light-dependent reactions utilize certain molecules to temporarily store the energy: These are referred to as energy carriers. The energy carriers that move energy from light-dependent reactions to light-independent reactions can be thought of as “full” because they are rich in energy. After the energy is released, the “empty” energy carriers return to the light-dependent reaction to obtain more energy. (Figure) illustrates the components inside the chloroplast where the light-dependent and light-independent reactions take place.

Click the link to learn more about photosynthesis.

Photosynthesis at the Grocery Store

Major grocery stores in the United States are organized into departments, such as dairy, meats, produce, bread, cereals, and so forth. Each aisle ((Figure)) contains hundreds, if not thousands, of different products for customers to buy and consume.

Although there is a large variety, each item ultimately can be linked back to photosynthesis. Meats and dairy link, because the animals were fed plant-based foods. The breads, cereals, and pastas come largely from starchy grains, which are the seeds of photosynthesis-dependent plants. What about desserts and drinks? All of these products contain sugar—sucrose is a plant product, a disaccharide, a carbohydrate molecule, which is built directly from photosynthesis. Moreover, many items are less obviously derived from plants: For instance, paper goods are generally plant products, and many plastics (abundant as products and packaging) are derived from “algae” (unicellular plant-like organisms, and cyanobacteria). Virtually every spice and flavoring in the spice aisle was produced by a plant as a leaf, root, bark, flower, fruit, or stem. Ultimately, photosynthesis connects to every meal and every food a person consumes.

Section Summary

The process of photosynthesis transformed life on Earth. By harnessing energy from the sun, the evolution of photosynthesis allowed living things access to enormous amounts of energy. Because of photosynthesis, living things gained access to sufficient energy that allowed them to build new structures and achieve the biodiversity evident today.

Only certain organisms (photoautotrophs), can perform photosynthesis they require the presence of chlorophyll, a specialized pigment that absorbs certain wavelengths of the visible spectrum and can capture energy from sunlight. Photosynthesis uses carbon dioxide and water to assemble carbohydrate molecules and release oxygen as a byproduct into the atmosphere. Eukaryotic autotrophs, such as plants and algae, have organelles called chloroplasts in which photosynthesis takes place, and starch accumulates. In prokaryotes, such as cyanobacteria, the process is less localized and occurs within folded membranes, extensions of the plasma membrane, and in the cytoplasm.

Visual Connection Questions

(Figure) On a hot, dry day, the guard cells of plants close their stomata to conserve water. What impact will this have on photosynthesis?

(Figure) Levels of carbon dioxide (a necessary photosynthetic substrate) will immediately fall. As a result, the rate of photosynthesis will be inhibited.

Review Questions

Which of the following components is not used by both plants and cyanobacteria to carry out photosynthesis?

What two main products result from photosynthesis?

  1. oxygen and carbon dioxide
  2. chlorophyll and oxygen
  3. sugars/carbohydrates and oxygen
  4. sugars/carbohydrates and carbon dioxide

In which compartment of the plant cell do the light-independent reactions of photosynthesis take place?

Which statement about thylakoids in eukaryotes is not correct?

  1. Thylakoids are assembled into stacks.
  2. Thylakoids exist as a maze of folded membranes.
  3. The space surrounding thylakoids is called stroma.
  4. Thylakoids contain chlorophyll.

Predict the end result if a chloroplast’s light-independent enzymes developed a mutation that prevented them from activating in response to light.

  1. GA3P accumulation
  2. ATP and NADPH accumulation
  3. Water accumulation
  4. Carbon dioxide depletion

How are the NADPH and GA3P molecules made during photosynthesis similar?

  1. They are both end products of photosynthesis.
  2. They are both substrates for photosynthesis.
  3. They are both produced from carbon dioxide.
  4. They both store energy in chemical bonds.

Critical Thinking Questions

What is the overall outcome of the light reactions in photosynthesis?

The outcome of light reactions in photosynthesis is the conversion of solar energy into chemical energy that the chloroplasts can use to do work (mostly anabolic production of carbohydrates from carbon dioxide).

Why are carnivores, such as lions, dependent on photosynthesis to survive?

Because lions eat animals that eat plants.

Why are energy carriers thought of as either “full” or “empty”?

The energy carriers that move from the light-dependent reaction to the light-independent one are “full” because they bring energy. After the energy is released, the “empty” energy carriers return to the light-dependent reaction to obtain more energy. There is not much actual movement involved. Both ATP and NADPH are produced in the stroma where they are also used and reconverted into ADP, Pi, and NADP + .

Describe how the grey wolf population would be impacted by a volcanic eruption that spewed a dense ash cloud that blocked sunlight in a section of Yellowstone National Park.

The grey wolves are apex predators in their food web, meaning they consume smaller prey animals and are not the prey of any other animal. Blocking sunlight would prevent the plants at the bottom of the food web from performing photosynthesis. This would kill many of the plants, reducing the food sources available to smaller animals in Yellowstone. A smaller prey animal population means that fewer wolves can survive in the area, and the population of grey wolves will decrease.

How does the closing of the stomata limit photosynthesis?

The stomata regulate the exchange of gases and water vapor between a leaf and its surrounding environment. When the stomata are closed, the water molecules cannot escape the leaf, but the leaf also cannot acquire new carbon dioxide molecules from the environment. This limits the light-independent reactions to only continuing until the carbon dioxide stores in the leaf are depleted.



Various starchless mutants have been used as test strains in search of higher lipid producers in many previous studies (Wang et al., 2009 Li et al., 2010a , b Work et al., 2010 Goodson et al., 2011 Siaut et al., 2011 Fan et al., 2012 Blaby et al., 2013 Goodenough et al., 2014 ). These studies also found that loss of starch synthesis and lower growth rates were strongly correlated, but the consequences on the individual PET reactions and carbon partitioning via pathways and into terminal products had not been examined systematically at the molecular level. Scheme 1 summarizes our data describing the molecular phenotypes arising from loss of AGPase in the Chlamydomonas starchless mutant sta6.

Starch biosynthesis (or glycogen biosynthesis in cyanobacteria) is essential for (at least) four reasons: (i) consuming the photosynthetically generated reductant (NADPH) and ATP (Kramer and Evans, 2011 ), (ii) providing a gluconeogenic route for replenishing CBB cycle intermediates, (iii) to produce a storage product with low osmotic potential to meet carbon and energy needs in the dark, and (iv) for photoprotection against oxidative damage. In the absence of starch biosynthesis arising from a non-functional AGPase, all of these functions are disrupted. Especially under high light, the primary effect is on the rate of CO2-dependent water oxidation, with sta6 having a 3.5-fold slower rate than cw15 (Figure 2a). This leads to a build-up in the reductant poise (NADPH/NADP + ), the extent of carbohydrate phosphorylation, and the level of adenylate nucleotides, the latter presumably in an attempt to compensate for the slower turnover rate (Scheme 1). Accumulation of several phosphorylated carbohydrates (Table 3 and Scheme 1) and the upper glycolysis intermediates glucose-1–phosphate, glucose-6–phosphate and fructose-6–phosphate occurs, and these do not drain efficiently to terminal sinks. This outcome, together with the 2.8-fold larger adenylate pool size, results in a corresponding decrease in the free inorganic phosphate (Pi) in the stroma of the chloroplast. In addition, the increased ATP would complex with Mg 2+ (Storer and Cornish-Bowden, 1976 ) leading to a decrease in the free Mg 2+ as well. As Mg 2+ and free Pi are required activators for RuBisCO and other CBB enzymes, allosteric down-regulation of RuBisCO dependent CO2 fixation activity is expected in sta6. This down-regulation appears to be directly responsible for the slowing of NADPH oxidation, and the ultimately slowing of all PET reactions as far upstream as water oxidation by PSII. The CBB cycle appears to be the main target affected by the AGPase mutation in sta6, as none of the individual PET enzyme fluxes exhibit inhibition relative to the cw15 control when measured in isolation. Our results with PET electron acceptors (Figure 3c) confirm that, like typical wild algal strains (Rochaix, 2011 ), re-oxidation of the PQ pool is retained in sta6 as the slowest kinetic step of the PET chain. sta6 uses mitochondrial respiration to help relieve aspects of cellular over-reduction (Figure 3b). By contrast, inhibition of mitochondrial respiration in cw15 leads to a comparatively insignificant effect on the OER. Thus, respiration appears to be critical for sustaining the low level of photosynthetic oxygen evolution in sta6. Consistent with this observation, P700 + kinetics are impaired when respiration is inhibited in sta6 (Johnson and Alric, 2012 ). For conditions under which sta6 is unable to maintain redox homeostasis, it does not depend on plastoquinol terminal oxidase or alternative oxidases but potentially uses the Mehler reaction or photorespiration to reduce O2 and remove the excess reductant. Thus alternative O2-consuming pathways become important for supporting light-induced ETR as redox valves in sta6.

The large fourfold increase in phosphoenolpyruvate concentration in sta6 is interesting as this molecule is known to function in another CO2 fixation pathway via phosphoenolpyruvate carboxylase, which may therefore become more important in sta6. Phosphoenolpyruvate carboxylase catalyzes the reaction that combines bicarbonate ( ) and phosphoenolpyruvate to form oxaloacetate. This is an anaplerotic reaction that is important for synthesis of amino acids (Ala, Asp, Glu). Phosphoenolpyruvate carboxylase-dependent carbon fixation may even exceed the amount of carbon fixed via the CBB cycle in some unicellular algae under nitrogen-limited conditions (Guy et al., 1989 ).

sta6 has smaller hexose phosphate pool sizes under low light, which may reflect the attenuation of photosynthesis, decreased carbon fixation, and/or re-direction of fixed carbon to the tricarboxylic acid cycle for protein synthesis. Under HL conditions, when increased levels of photosynthate enter the central metabolism, the tricarboxylic acid cycle appears to become saturated (Table 3), resulting in accumulation of hexose phosphate metabolites. Determination of the precise underpinnings of these phenotypes requires additional experimentation, and metabolic flux analysis is required to obtain a more informed understanding of the observed dynamic re-direction of metabolites.

Even though over-reduction of the PET chain is relieved in part by mitochondrial respiration in sta6 (Figure 3b), prolonged exposure to high light leads to accelerated photoinhibition relative to cw15 (Figure 5b), indicating that starch production is an important safeguard against photodamage. The light-driven accumulation of excess NADPH and reduced ferredoxin lead to formation of reactive oxygen species in many aquatic phototrophs and plants. A similar phenotype was observed in several other characterized AGPase mutants (Sun et al., 1999 Suzuki et al., 2010 Grundel et al., 2012 ). As starch is the least energy-intensive terminal product among starch, proteins and lipids, it may serve as an energy storage buffer to protect against light energy fluctuations and to enable biosynthesis of these more energy-intensive biopolymers during low light periods, similar to that postulated in higher plants (Caspar et al., 1985 Stitt and Quick, 1989 Ludewig et al., 1998 Geigenberger, 2011 Weise et al., 2011 ).

Carbon that enters the central metabolism from the CBB cycle may take two possible routes: the upper gluconeogenesis pathway leading into C6 and C5 carbohydrates, or the lower glycolytic pathway leading to acetyl CoA that either enters the tricarboxylic acid cycle to synthesize amino acids or is converted to malonyl CoA to synthesize lipids. Several groups have reported increased lipid yields in sta6 after switching cells to a nitrogen-deficient growth medium (Wang et al., 2009 Li et al., 2010a , b Work et al., 2010 Goodson et al., 2011 Siaut et al., 2011 Fan et al., 2012 ). Increased lipid accumulation is not observed under nutrient-replete, photoautotrophic conditions in sta6 instead growth is stunted. However, there is a major increase in both acetyl CoA and malonyl CoA levels in sta6. Malonyl CoA formation is catalyzed by acetyl CoA carboxylase (ACCase) through an ATP-dependent carboxylation of biotin that transfers a carboxyl group to acetyl CoA (Berg et al., 2002 ). The ACCase reaction is considered to be the committed step in fatty acid synthesis. It is regulated by reversible phosphorylation catalyzed by an AMP-dependent protein kinase (Berg et al., 2002 ). ACCase is inhibited by phosphorylation, while the unphosphorylated form has carboxylase activity. AMP-dependent protein kinase itself acts as an adenylate nucleotide sensor, being activated by high AMP levels and inhibited by high ATP levels (Berg et al., 2002 Hardie and Pan, 2002 ). As such, the kinase activity becomes self-limiting at high ATP concentrations. Accumulation of malonyl CoA in sta6 indicates a reversal of inhibition of ACCase relative to cw15. Given that ATP accumulates in sta6 (Table 3), we predict that the higher ATP content probably inhibits AMP-dependent protein kinase, and, in turn, enhances the carboxylase activity of ACCase. Thus, simple over-expression of ACCase may not be enough to enhance ACCase activity, but instead needs to occur in parallel with reducing the level of phosphorylation of ACCase, which is achieved by suppression of AMP-dependent protein kinase phosphorylation activity. Taken together, the higher ATP accumulation in sta6 cells metabolically poises it for higher levels of fatty acyl biosynthesis, as indicated by elevated levels of acetyl CoA and especially malonyl CoA, but downstream blockage in the fatty acid synthesis complex putatively prevents utilization. Our data show that factors beyond availability of the precursor (malonyl CoA) control the flux into lipid biosynthesis. Under photoautotrophic nutrient-replete conditions, downstream enzymatic reactions, possibly involving the fatty acid synthase complex or glycerolipid biosynthetic enzymes, appear to be limiting lipid biosynthesis. If the flux through these enzymes were increased, sta6 may be able to utilize the substantially elevated pool of malonyl CoA. Transcriptomic studies of C. reinhardtii have shown a significant increase in the content of acyl transferases such as acyl CoA/diacylglycerol acyltransferase (DGAT) and phospholipid/diacylglycerol acyltransferase, and suggested the involvement of transcription factor NRR1 in potentially regulating the expression of certain diacylglycerol acyltransferase genes and triacylglycerol accumulation (Boyle et al., 2012 ). The activities of these enzymes in particular may have to be increased to achieve improved lipid yields from malonyl CoA in sta6.

In conclusion, our data indicate that, in C. reinhardtii, starch biosynthesis plays a critical role in regulating multiple functions, largely through accumulation/utilization of redox and adenylate cofactors. Future research efforts are required to examine mechanisms to effectively leverage the greatly increased malonyl CoA levels for enhanced lipid biosynthesis, which, if successful, may allow higher NADPH re-oxidation rates and restore photosynthetic productivities.


J.W. BRADBEER , G. MONTES , in Light and Plant Development , 1976

Control of Chloroplast Development by Light Quality

When seedlings are grown under continuous far-red irradiation, they resemble seedlings grown in continuous white light in most of their visible characteristics except that they remain etiolated and accumulate little chlorophyll ( Mohr, 1972 ). Examination of the fine structure of bean seedlings grown under continuous far-red irradiation shows that they do not contain normal chloroplasts, and the plastids have parallel thylakoid sheets with no grana and only a small amount of thylakoid appression where adjacent thylakoid sheets overlap ( De Greef, Butler and Roth, 1971 ). In mustard cotyledons, prolonged far-red irradiation results in plastids with crystalline prolamellar bodies and parallel thylakoid sheets with a substantial amount of thylakoid appression, to give what may be described as paired primary thylakoids rather than grana ( Häcker, 1967 Kasemir, Bergfeld and Mohr, 1975 ). The latter workers also showed that in etioplast development in the cotyledons of dark-grown mustard the prolamellar bodies at first possessed a crystalline form which subsequently was lost. However, crystallisation of the prolamellar body occurred in response to either prolonged far-red or short-red irradiation, and to some extent in response to short far-red irradiation, and was inferred to result from the action of phytochrome. Berry and Smith (1971) found that short red-light treatments induced prolamellar body crystallisation in dark-grown barley leaves but as there was no far-red induced reversal they inferred that phytochrome was not involved. In the primary leaves of dark-grown Phaseolus, the prolamellar bodies retain their crystalline form, at least up to 30 days of dark growth, when senescence and death intervene ( Bradbeer et al., 1974a ). Thus we have not been able to observe irradiation-induced prolamellar body crystallisation in bean, but it is possible that irradiation at an early stage of etioplast development might induce such a crystallisation.

In angiosperms, normal chloroplasts have so far been found only in plants which have been exposed to substantial periods of irradiation with white light. Since chloroplast development is clearly not wholly controlled by phytochrome, it is evident that the process requires both phytochrome and other photoreceptors.

The complexity of a situation involving more than one photoreceptor, together with the time-consuming nature of the quantitative investigation of fine structure which will be required to resolve the situation, has resulted in very slow progress in this area. As an initial investigation, we studied the effects of short exposures of dark-grown beans to irradiation of different wavelengths. The following broad bandwidth illumination treatments were used.

Blue: 20 min exposure to 50 μW cm −2 , equivalent to 230 nE cm −2 per irradiation peak transmission, 450 nm bandwidth at half peak transmission, 47 nm.

Red: 5 min exposure to 178 μW cm −2 , equivalent to 280 nE cm −2 per irradiation peak transmission, 634 nm bandwidth at half peak transmission, 57 nm.

Far-red: 20 min exposure to the following distribution of radiation: <700 nm of 6 μW cm −2 (40 nE cm −2 ), 700–750 nm of 32 μW cm −2 (230 nE cm −2 ), 750–800 nm of 112 μW cm −2 (870 nE cm −2 ).

White: 5 min exposure to 900 μW cm −2 , equivalent to 1300 nE cm −2 , from Ekco ‘Double Light’ fluorescent tubes.

Dark-grown beans were exposed to these irradiations, supplied either singly or in certain combinations, once on each of days 12, 13 and 14. They were maintained in darkness between the irradiation treatments and fixed for analysis on day 15, exactly 24 h after the preceding irradiation ( Bradbeer, 1971 Bradbeer et al., 1974c ). The results of the short illumination treatments may be compared with those of investigations of the effects of continuous white light on chloroplast development ( Bradbeer, 1969 Bradbeer et al., 1969 , 1974b ).

Table 15.1 shows the effects of the various short irradiation treatments on several parameters of leaf and plastid development. For comparison, the effects of 3 days' continuous irradiation with white light (1.6 mW cm −2 ) on 12-day-old dark-grown beans are also included.

Table 15.1 . The ‘slow’ effects of the irradiation of 12-day-old dark-grown Phaseolus vulgaris L. cv. Alabaster seedlings Short irradiation treatments given on days 12, 13 and 14. Leaves analysed after 15 days' dark growth, 24 h after the preceding short light treatment.

TreatmentPer primary leaf
Plastid diameter (μm)Leaf area (mm 2 )Fresh weight (mg)Dry weight (mg)Total protein (mg)10 −6 × cell number10 −7 × plastid numberPlastid membrane (cm 2 )
12 days dark2.3300346.52.11523189
15 days dark2.8309376.82.31528327
5 min red daily3.7708508.42.82151757
20 min far-red daily2.9354417.42.41735391
5 min red/20 min far-red daily2.7321417.22.21627213
20 min blue daily2.8344387.22.31628289
5 min red/20 min blue daily3.1659518.73.12341503
5 min white daily3.81072528.73.32458729
Continuous white light4.3146115516.76.523582000 1

None of the measurements recorded in Table 15.1 show any difference between the short blue irradiation and the dark control. Both the increases in leaf area and in fresh and dry weights satisfied the operational requirements for control by Pfr, being promoted by red, reversed by brief far-red and scarcely affected by brief far-red light alone. A similar observation was reported by Downs (1955) . Short white irradiation was more effective than red in increasing leaf area, apparently by promoting the development of intercellular spaces. It is possible that this latter aspect of leaf development is not a phytochrome response. In continuous white irradiation, the leaf area was close to that found after short white irradiations but the fresh and dry weights were much higher, possibly resulting from the photosynthetic activity of the leaf in continuous light increasing the dry matter content of the leaf. There was some light-induced cell division which was clearly under the operational control of phytochrome without any evidence of the participation of another photoreceptor. Table 15.1 also shows that plastid division, plastid expansion and the formation of plastid membrane were all under the operational control of phytochrome but that when red irradiation was followed by blue there was a statistically significant reduction of the red-induced promotion. This result differs somewhat from that of Possingham (1973) , who found that chloroplast replication in cultured leaf discs of spinach required either a high intensity of continuous white light (6.5 mW cm −2 ) or high irradiations from red or blue laser light. On the other hand, Pfr has been reported to control chloroplast replication in the germinating spores of Polytrichum ( Kass and Paolillo, 1974 ). Kasemir, Bergfeld and Mohr (1975) have shown that plastid expansion is controlled by Pfr. As the plastid membrane content increased much more under continuous illumination than under short illumination, it is evidently under the control of another photoreceptor in addition to phytochrome. The total leaf protein content was also under the operational control of phytochrome but the red-induced increase was stimulated further by following red with blue irradiation. In this case, continuous illumination was more effective than short illumination. Table 15.2 summarises our conclusions about the slow effects of phytochrome.

Table 15.2 . Summary of photoreceptors considered to be involved in various ‘slow’ reactions in leaf and chloroplast development in Phaseolus vulgaris L. cv. Alabaster

ReactionPfrContinuous white irradiation more effective than short red treatmentRed/blue interaction
Leaf expansion++ 1 0
Increase in leaf dry weight++0
Cell division+00
Plastid division+0
Plastid expansion++
Plastid membrane formation++
Grana formation0+0
Accumulation of leaf protein+++

In an attempt to elucidate the mechanism of some fine structural changes not discussed in this chapter, an investigation of the ‘rapid’ effects of various irradiation treatments was carried out by taking samples at intervals during the 3-h period immediately after the irradiation of 12-day-old dark-grown beans. In addition, a sample was fixed after 24 h. Although the results of these latter experiments have been discussed in detail elsewhere ( Bradbeer et al., 1974c ), they are summarised in Table 15.3 .

Table 15.3 . The ‘rapid’ effects on etioplast fine structure of the exposure of 12-day-old dark-grown beans to short irradiation treatments

TreatmentPercentage of prolamellar bodies transformed within 1 h of illuminationMaximum percentage loss of prolamellar body volume within 3 h of illuminationOccurrence of prolamellar body recondensation within 3 h of illuminationMaximum percentage change of area of thylakoids within 3 h of illumination
5 min red100900+23
20 min far-red10090+−19
5 min red/20 min far-red10075++ 35
20 min blue3755+−31
5 min red/20 min blue1001000+23
5 min white100800+44

All of the irradiation treatments except blue gave complete prolamellar body transformation from the crystalline state to the reacted state within 30–40 min of the commencement of illumination. Continuous white irradiation gave a similar effect. The average volume of prolamellar body per plastid also fell in response to all of the irradiations, although in this case blue again was least effective. Within 3 h of the commencement of irradiation, prolamellar body recondensation had commenced in the case of the far-red, the red/far-red and the blue treatments. This result suggests that prolamellar body recondensation may be inhibited by Pfr while, in contrast, the crystallisation of the mustard prolamellar body has been reported to require Pfr ( Kasemir, Bergfeld and Mohr, 1975 ). The area of the thylakoids increased in response to all irradiation treatments which included a red component and fell in response to all irradiation treatments which did not include red (far-red and blue). This effect indicates that thylakoid formation is dependent on a red-absorbing photoreceptor other than phytochrome. Kasemir, Bergfeld and Mohr (1975) suggest that the formation of primary thylakoids may result from the conversion of protochlorophyllide to chlorophyllide.

The rapid responses in the etioplast which seem to be phytochrome controlled are prolamellar body recondensation ( Table 15.3 ), the crystallisation of the prolamellar body ( Kasemir, Bergfeld and Mohr, 1975 ), the stimulation of the Shibata shift ( Jabben and Mohr, 1975 ) and the release of gibberellin-like substances from etioplast membranes (Evans, Chapter 10 ). Of these, at least the first three are presumably located in the prolamellar body, but only the first two are fine-structural changes and only the first one has been seen in etiolated beans, which already possess crystalline prolamellar bodies.

Wellburn and Wellburn ( 1973a ) reported a ‘rapid’ effect of red light (which fulfilled the operational criteria for phytochrome) on the fine structure of etioplasts both in vivo and in vitro. However, the result was obtained by the application of their index of chloroplast development, which represents the transformation and loss of the prolamellar body in one dimension and the elaboration of the thylakoids in the other dimension ( Wellburn and Wellburn, 1973b ). It appears to us that the measurement of two distinct processes on the same scale is both liable to give misleading results and certain to fail to define the exact nature of the photoresponses. Although prolamellar body loss may well induce further thylakoid development ( Bradbeer et al., 1974b ), it is clear from our data ( Table 15.3 and Bradbeer et al., 1974c ) that these two processes are separate and are probably controlled differently. Henningsen (1967) found that the process of vesicle dispersal was controlled by a photoreceptor with an action spectrum consisting of a sharp peak at 450 nm. Vesicle dispersal is an artefact resulting from the use of permanganate fixation in electron microscopy but the stage is probably equivalent to prolamellar body dispersal which is seen in glutaraldehyde/OsO4-fixed material. Unfortunately, Henningsen's experiments have not been repeated with glutaraldehyde/OsO4 fixation and the nature of the response that he studied has not been resolved. At least the process does not seem to involve phytochrome. The investigation of the ‘rapid’ effects of light on etioplasts clearly requires further investigation.

8.21: Light-Independent Reactions - Biology

A. Two Pathways
1. Two electron pathways operate in the thylakoid membrane: the noncyclic pathway and the cyclic pathway.
2. Both pathways produce ATP but only the noncyclic pathway also produces NADPH.
3. ATP production during photosynthesis is sometimes called photophosphorylation therefore these pathways are also known as cyclic and noncyclic photophosphorylation.

B. Noncyclic Electron Pathway (*SPLITS WATER, PRODUCES NADPH & ATP)

1. This pathway occurs in the thylakoid membranes and requires participation of two light-gathering units: photosystem I (PS I) and photosystem II (PS II).
2. A photosystem is a photosynthetic unit comprised of a pigment complex and electron acceptor solar energy is absorbed and high-energy electrons are generated.
3. Each photosystem has a pigment complex composed of green chlorophyll a and chlorophyll b molecules and orange and yellow accessory pigments (e.g., carotenoid pigments).
4. Absorbed energy is passed from one pigment molecule to another until concentrated in reaction-center chlorophyll a.
5. Electrons in reaction-center chlorophyll a become excited they escape to electron-acceptor molecule.
6. The noncyclic pathway begins with PSII electrons move from H2O through PS II to PS I and then on to NADP+.
7. The PS II pigment complex absorbs solar energy high-energy electrons (e-) leave the reaction-center chlorophyll a molecule.
8. PS II takes replacement electrons from H2O, which splits, releasing O2 and H+ ions:
9. Oxygen is released as oxygen gas (O2).
10. The H+ ions temporarily stay within the thylakoid space and contribute to a H+ ion gradient.
11. As H+ flow down electrochemical gradient through ATP synthase complexes, chemiosmosis occurs.
12. Low-energy electrons leaving the electron transport system enter PS I.
13. When the PS I pigment complex absorbs solar energy, high-energy electrons leave reaction-center chlorophyll a and are captured by an electron acceptor.
14. The electron acceptor passes them on to NADP+.
15. NADP+ takes on an H+ to become NADPH: NADP+ + 2 e- + H+  NADPH.
16. NADPH and ATP produced by noncyclic flow electrons in thylakoid membrane are used by enzymes in stroma during light-independent reactions.

C. Cyclic Electron Pathway
1. The cyclic electron pathway begins when the PS I antenna complex absorbs solar energy.
2. High-energy electrons leave PS I reaction-center chlorophyll a molecule.
3. Before they return, the electrons enter and travel down an electron transport system.

a. Electrons pass from a higher to a lower energy level.
b. Energy released is stored in form of a hydrogen (H+) gradient.
c. When hydrogen ions flow down their electrochemical gradient through ATP synthase complexes, ATP production occurs.
d. Because the electrons return to PSI rather than move on to NADP+, this is why it is called cyclic and also why no NADPH is produced.

D. ATP Production (chemiosmosis)
1. The thylakoid space acts as a reservoir for H+ ions each time H2O is split, two H+ remain.
2. Electrons move carrier-to-carrier, giving up energy used to pump H+ from the stroma into the thylakoid space.
3. Flow of H+ from high to low concentration across thylakoid membrane provides energy to produce ATP from ADP + P by using an ATP synthase enzyme

**Now is a good time to look at the various animations of these processes. The trick is to VISUALIZE them*

Generating an Energy Molecule: ATP

In the light-dependent reactions, energy absorbed by sunlight is stored by two types of energy-carrier molecules: ATP and NADPH. The energy that these molecules carry is stored in a bond that holds a single atom to the molecule. For ATP, it is a phosphate atom, and for NADPH, it is a hydrogen atom. Recall that NADH was a similar molecule that carried energy in the mitochondrion from the citric acid cycle to the electron transport chain. When these molecules release energy into the Calvin cycle, they each lose atoms to become the lower-energy molecules ADP and NADP + .

The buildup of hydrogen ions in the thylakoid space forms an electrochemical gradient because of the difference in the concentration of protons (H + ) and the difference in the charge across the membrane that they create. This potential energy is harvested and stored as chemical energy in ATP through chemiosmosis, the movement of hydrogen ions down their electrochemical gradient through the transmembrane enzyme ATP synthase, just as in the mitochondrion.

The hydrogen ions are allowed to pass through the thylakoid membrane through an embedded protein complex called ATP synthase. This same protein generated ATP from ADP in the mitochondrion. The energy generated by the hydrogen ion stream allows ATP synthase to attach a third phosphate to ADP, which forms a molecule of ATP in a process called photophosphorylation. The flow of hydrogen ions through ATP synthase is called chemiosmosis (just like in cellular respiration), because the ions move from an area of high to low concentration through a semi-permeable structure.

Biohydrogen from Microalgae, Uniting Energy, Life, and Green Future

3.1 How do Microalgae Produce Biohydrogen?

Microalgae are photosynthetic microorganisms that can harvest solar energy by special molecules (chlorophyll, carotenoid, phycobilin) to fix CO2 and produce carbohydrates. Photosynthesis can be classified basically into two stages: (1) the light-dependent stage and (2) the light-independent (dark) stage. Light reactions takes place in the thylakoid membranes that contain light-harvesting antenna, photosystem I (PSI), photosystem II (PSII), cytochrome b6f, and ATP synthase complexes. Photosystems also have reaction centers P700 (PSI) and P680 (PSII) where specific wavelengths of light can be harvested. The light reactions are responsible for the supply of NADPH2 and ATP to fix inorganic carbon during the Calvin–Benson Cycle of the dark stage ( Walker, 2009 Antal et al., 2011 Masojidek et al., 2013 ). On the other hand, photorespiration is a light-independent reaction that converts organic carbon back to CO2. During respiration, the CO2-fixing Rubisco, which actually has the ability to bind to both O2 and CO2, acts as an oxygenase. Rubisco fixes according to the partial pressure changes of CO2 and O2 if the pressure is in favor of CO2 it will fix CO2, and if not, it fixes O2. Photorespiration is an unfavorable process that decreases the efficiency of photosynthesis in microalgae, because it consumes energy but does not produce any sugars. But because it can decrease oxygen levels in the culture, it can be important for hydrogen production ( Miyake, 1998 Zhu et al., 2008 Masojidek et al., 2013 Oncel, 2013 Torzillo and Seibert, 2013 ). This prelude is the first step to understand metabolic activities for biohydrogen generation.

Hydrogen plays an important role in photosynthesis as being the vehicle in the photosynthetic transport grid where plants and microalgae can utilize water as the source of hydrogen ( Antal et al., 2011 Polander and Barry, 2012 ). Different routes that are active in the hydrogen evolution process of microalgae are related to the dissipation of excess electrons of the electron transport chain when the main electron acceptors CO2 and O2 are absent or the other processes like Calvin cycle, photorespiration, and Mehler reactions used as sinks are blocked ( Ghysels and Franck, 2010 Antal et al., 2011 ). The key of these routes is the transfer of electrons to hydrogen evolving enzymes, whether supplied directly from water splitting or indirectly from organic materials such as starch ( Kruse and Hankamer, 2010 ).

The first route is the direct biophotolysis of water into hydrogen and oxygen where the water-splitting PSII and ferredoxin-reducing PSI act cooperatively. The second route is the indirect biophotolysis where the electrons from glycolysis are transferred to the linear electron transport chain and utilized only by a PSI active hydrogen evolution pathway. A third route other than these light-dependent routes is the dark fermentation of the decarboxylated pyruvate from glycolysis by pyruvate ferredoxin oxidoreductase ( Beer et al., 2009 Maness et al., 2009 Ghysels and Franck, 2010 Torzillo et al., 2014 ).

Applications that can show important enhancement with regard to biohydrogen productivities in microalgae ( Lopes Pinto et al., 2002 Mathews and Wang, 2009 Oncel and Kose, 2014 Torzillo et al., 2014 ) include the proper changes in several culture parameters such as adjusting salinity, percentages of sparged gasses (CO2, Ar), nutrient levels, illumination patterns as well as partial pressure of head space.


In chemistry, many reactions depend on the absorption of photons to provide the energy needed to overcome the activation energy barrier and hence can be labelled light-dependent. Such reactions range from the silver halide reactions used in photographic film to the creation and destruction of ozone in the upper atmosphere. This article discusses a specific subset of these, the series of light-dependent reactions related to photosynthesis in living organisms.

The reaction center is in the thylakoid membrane. It transfers light energy to a dimer of chlorophyll pigment molecules near the periplasmic (or thylakoid lumen) side of the membrane. This dimer is called a special pair because of its fundamental role in photosynthesis. This special pair is slightly different in PSI and PSII reaction center. In PSII, it absorbs photons with a wavelength of 680 nm, and it is therefore called P680. In PSI, it absorbs photons at 700 nm, and it is called P700. In bacteria, the special pair is called P760, P840, P870, or P960. "P" here means pigment, and the number following it is the wavelength of light absorbed.

If an electron of the special pair in the reaction center becomes excited, it cannot transfer this energy to another pigment using resonance energy transfer. In normal circumstances, the electron should return to the ground state, but, because the reaction center is arranged so that a suitable electron acceptor is nearby, the excited electron can move from the initial molecule to the acceptor. This process results in the formation of a positive charge on the special pair (due to the loss of an electron) and a negative charge on the acceptor and is, hence, referred to as photoinduced charge separation. In other words, electrons in pigment molecules can exist at specific energy levels. Under normal circumstances, they exist at the lowest possible energy level they can. However, if there is enough energy to move them into the next energy level, they can absorb that energy and occupy that higher energy level. The light they absorb contains the necessary amount of energy needed to push them into the next level. Any light that does not have enough or has too much energy cannot be absorbed and is reflected. The electron in the higher energy level, however, does not want to be there the electron is unstable and must return to its normal lower energy level. To do this, it must release the energy that has put it into the higher energy state to begin with. This can happen various ways. The extra energy can be converted into molecular motion and lost as heat. Some of the extra energy can be lost as heat energy, while the rest is lost as light. (This re-emission of light energy is called fluorescence.) The energy, but not the e- itself, can be passed onto another molecule. (This is called resonance.) The energy and the e- can be transferred to another molecule. Plant pigments usually utilize the last two of these reactions to convert the sun's energy into their own.

This initial charge separation occurs in less than 10 picoseconds (10 −11 seconds). In their high-energy states, the special pigment and the acceptor could undergo charge recombination that is, the electron on the acceptor could move back to neutralize the positive charge on the special pair. Its return to the special pair would waste a valuable high-energy electron and simply convert the absorbed light energy into heat. In the case of PSII, this backflow of electrons can produce reactive oxygen species leading to photoinhibition. [1] [2] Three factors in the structure of the reaction center work together to suppress charge recombination nearly completely.

  • Another electron acceptor is less than 10 Å away from the first acceptor, and so the electron is rapidly transferred farther away from the special pair.
  • An electron donor is less than 10 Å away from the special pair, and so the positive charge is neutralized by the transfer of another electron
  • The electron transfer back from the electron acceptor to the positively charged special pair is especially slow. The rate of an electron transfer reaction increases with its thermodynamic favorability up to a point and then decreases. The back transfer is so favourable that it takes place in the inverted region where electron-transfer rates become slower. [1]

Thus, electron transfer proceeds efficiently from the first electron acceptor to the next, creating an electron transport chain that ends if it has reached NADPH.

The photosynthesis process in chloroplasts begins when an electron of P680 of PSII attains a higher-energy level. This energy is used to reduce a chain of electron acceptors that have subsequently lowered redox-potentials. This chain of electron acceptors is known as an electron transport chain. When this chain reaches PS I, an electron is again excited, creating a high redox-potential. The electron transport chain of photosynthesis is often put in a diagram called the z-scheme, because the redox diagram from P680 to P700 resembles the letter Z. [3]

The final product of PSII is plastoquinol, a mobile electron carrier in the membrane. Plastoquinol transfers the electron from PSII to the proton pump, cytochrome b6f. The ultimate electron donor of PSII is water. Cytochrome b6f proceeds the electron chain to PSI through plastocyanin molecules. PSI is able to continue the electron transfer in two different ways. It can transfer the electrons either to plastoquinol again, creating a cyclic electron flow, or to an enzyme called FNR (Ferredoxin—NADP(+) reductase), creating a non-cyclic electron flow. PSI releases FNR into the stroma, where it reduces NADP +

Activities of the electron transport chain, especially from cytochrome b6f, lead to pumping of protons from the stroma to the lumen. The resulting transmembrane proton gradient is used to make ATP via ATP synthase.

The overall process of the photosynthetic electron transport chain in chloroplasts is:

2 O → PS II → plastoquinone → cyt b6f → plastocyanin → PS I → NADPH

Photosystem II Edit

PS II is extremely complex, a highly organized transmembrane structure that contains a water-splitting complex, chlorophylls and carotenoid pigments, a reaction center (P680), pheophytin (a pigment similar to chlorophyll), and two quinones. It uses the energy of sunlight to transfer electrons from water to a mobile electron carrier in the membrane called plastoquinone:

2 O
P680P680 *plastoquinone

Plastoquinone, in turn, transfers electrons to cyt b6f, which feeds them into PS I.

The water-splitting complex Edit

The step H
2 O → P680
is performed by a poorly understood structure embedded within PS II called the water-splitting complex or the oxygen-evolving complex. It catalyzes a reaction that splits water into electrons, protons and oxygen:

2 H
2 O
4H + + 4e − + O

The actual steps of the above reaction are running in the following way (Dolai's diagram of S-states): (I) 2 H
2 O (monoxide) (II) OH. H
2 O (hydroxide) (III) H
2 O
2 (peroxide) (IV) HO
2 (super oxide)(V) O
2 (di-oxygen). [ citation needed ] (Dolai's mechanism)

The electrons are transferred to special chlorophyll molecules (embedded in PS II) that are promoted to a higher-energy state by the energy of photons.

The reaction center Edit

The excitation P680 → P680 * of the reaction center pigment P680 occurs here. These special chlorophyll molecules embedded in PS II absorb the energy of photons, with maximal absorption at 680 nm. Electrons within these molecules are promoted to a higher-energy state. This is one of two core processes in photosynthesis, and it occurs with astonishing efficiency (greater than 90%) because, in addition to direct excitation by light at 680 nm, the energy of light first harvested by antenna proteins at other wavelengths in the light-harvesting system is also transferred to these special chlorophyll molecules.

This is followed by the step P680 * → pheophytin, and then on to plastoquinone, which occurs within the reaction center of PS II. High-energy electrons are transferred to plastoquinone before it subsequently picks up two protons to become plastoquinol. Plastoquinol is then released into the membrane as a mobile electron carrier.

This is the second core process in photosynthesis. The initial stages occur within picoseconds, with an efficiency of 100%. The seemingly impossible efficiency is due to the precise positioning of molecules within the reaction center. This is a solid-state process, not a chemical reaction. It occurs within an essentially crystalline environment created by the macromolecular structure of PS II. The usual rules of chemistry (which involve random collisions and random energy distributions) do not apply in solid-state environments.

Link of water-splitting complex and chlorophyll excitation Edit

When the chlorophyll passes the electron to pheophytin, it obtains an electron from P680 * . In turn, P680 * can oxidize the Z (or YZ) molecule. Once oxidized, the Z molecule can derive electrons from the oxygen-evolving complex (OEC). [4] Dolai's S-state diagrams show the reactions of water splitting in the oxygen-evolving complex.

Summary Edit

PS II is a transmembrane structure found in all chloroplasts. It splits water into electrons, protons and molecular oxygen. The electrons are transferred to plastoquinone, which carries them to a proton pump. Molecular oxygen is released into the atmosphere.

The emergence of such an incredibly complex structure, a macromolecule that converts the energy of sunlight into potentially useful work with efficiencies that are impossible in ordinary experience, seems almost magical at first glance. Thus, it is of considerable interest that, in essence, the same structure is found in purple bacteria.

Cytochrome b6f Edit

PS II and PS I are connected by a transmembrane proton pump, cytochrome b6f complex (plastoquinol—plastocyanin reductase EC Electrons from PS II are carried by plastoquinol to cyt b6f, where they are removed in a stepwise fashion (reforming plastoquinone) and transferred to a water-soluble electron carrier called plastocyanin. This redox process is coupled to the pumping of four protons across the membrane. The resulting proton gradient (together with the proton gradient produced by the water-splitting complex in PS II) is used to make ATP via ATP synthase.

The structure and function of cytochrome b6f (in chloroplasts) is very similar to cytochrome bc1 (Complex III in mitochondria). Both are transmembrane structures that remove electrons from a mobile, lipid-soluble electron carrier (plastoquinone in chloroplasts ubiquinone in mitochondria) and transfer them to a mobile, water-soluble electron carrier (plastocyanin in chloroplasts cytochrome c in mitochondria). Both are proton pumps that produce a transmembrane proton gradient. In fact, cytochrome b6 and subunit IV are homologous to mitochondrial cytochrome b [5] and the Rieske iron-sulfur proteins of the two complexes are homologous. [6] However, cytochrome f and cytochrome c1 are not homologous. [7]

Photosystem I Edit

PS I accepts electrons from plastocyanin and transfers them either to NADPH (noncyclic electron transport) or back to cytochrome b6f (cyclic electron transport):

PS I, like PS II, is a complex, highly organized transmembrane structure that contains antenna chlorophylls, a reaction center (P700), phylloquinone, and a number of iron-sulfur proteins that serve as intermediate redox carriers.

The light-harvesting system of PS I uses multiple copies of the same transmembrane proteins used by PS II. The energy of absorbed light (in the form of delocalized, high-energy electrons) is funneled into the reaction center, where it excites special chlorophyll molecules (P700, maximum light absorption at 700 nm) to a higher energy level. The process occurs with astonishingly high efficiency.

Electrons are removed from excited chlorophyll molecules and transferred through a series of intermediate carriers to ferredoxin, a water-soluble electron carrier. As in PS II, this is a solid-state process that operates with 100% efficiency.

There are two different pathways of electron transport in PS I. In noncyclic electron transport, ferredoxin carries the electron to the enzyme ferredoxin NADP +
reductase (FNR) that reduces NADP +
to NADPH. In cyclic electron transport, electrons from ferredoxin are transferred (via plastoquinone) to a proton pump, cytochrome b6f. They are then returned (via plastocyanin) to P700.

NADPH and ATP are used to synthesize organic molecules from CO
2 . The ratio of NADPH to ATP production can be adjusted by adjusting the balance between cyclic and noncyclic electron transport.

It is noteworthy that PS I closely resembles photosynthetic structures found in green sulfur bacteria, just as PS II resembles structures found in purple bacteria.

PS II, PS I, and cytochrome b6f are found in chloroplasts. All plants and all photosynthetic algae contain chloroplasts, which produce NADPH and ATP by the mechanisms described above. In essence, the same transmembrane structures are also found in cyanobacteria.

Unlike plants and algae, cyanobacteria are prokaryotes. They do not contain chloroplasts. Rather, they bear a striking resemblance to chloroplasts themselves. This suggests that organisms resembling cyanobacteria were the evolutionary precursors of chloroplasts. One imagines primitive eukaryotic cells taking up cyanobacteria as intracellular symbionts in a process known as endosymbiosis.

Cyanobacteria Edit

Cyanobacteria contain both PS I and PS II. Their light-harvesting system is different from that found in plants (they use phycobilins, rather than chlorophylls, as antenna pigments), but their electron transport chain

is, in essence, the same as the electron transport chain in chloroplasts. The mobile water-soluble electron carrier is cytochrome c6 in cyanobacteria, plastocyanin in plants.

Cyanobacteria can also synthesize ATP by oxidative phosphorylation, in the manner of other bacteria. The electron transport chain is

where the mobile electron carriers are plastoquinone and cytochrome c6, while the proton pumps are NADH dehydrogenase, cyt b6f and cytochrome aa3 (member of the COX3 family).

Cyanobacteria are the only bacteria that produce oxygen during photosynthesis. Earth's primordial atmosphere was anoxic. Organisms like cyanobacteria produced our present-day oxygen-containing atmosphere.

The other two major groups of photosynthetic bacteria, purple bacteria and green sulfur bacteria, contain only a single photosystem and do not produce oxygen.

Purple bacteria Edit

Purple bacteria contain a single photosystem that is structurally related to PS II in cyanobacteria and chloroplasts:

P870 → P870 * → ubiquinone → cyt bc1 → cyt c2 → P870

This is a cyclic process in which electrons are removed from an excited chlorophyll molecule (bacteriochlorophyll P870), passed through an electron transport chain to a proton pump (cytochrome bc1 complex similar to the chloroplastic one), and then returned to the chlorophyll molecule. The result is a proton gradient, which is used to make ATP via ATP synthase. As in cyanobacteria and chloroplasts, this is a solid-state process that depends on the precise orientation of various functional groups within a complex transmembrane macromolecular structure.

To make NADPH, purple bacteria use an external electron donor (hydrogen, hydrogen sulfide, sulfur, sulfite, or organic molecules such as succinate and lactate) to feed electrons into a reverse electron transport chain.

Green sulfur bacteria Edit

Green sulfur bacteria contain a photosystem that is analogous to PS I in chloroplasts:

There are two pathways of electron transfer. In cyclic electron transfer, electrons are removed from an excited chlorophyll molecule, passed through an electron transport chain to a proton pump, and then returned to the chlorophyll. The mobile electron carriers are, as usual, a lipid-soluble quinone and a water-soluble cytochrome. The resulting proton gradient is used to make ATP.

In noncyclic electron transfer, electrons are removed from an excited chlorophyll molecule and used to reduce NAD + to NADH. The electrons removed from P840 must be replaced. This is accomplished by removing electrons from H
2 S , which is oxidized to sulfur (hence the name "green sulfur bacteria").

Purple bacteria and green sulfur bacteria occupy relatively minor ecological niches in the present day biosphere. They are of interest because of their importance in precambrian ecologies, and because their methods of photosynthesis were the likely evolutionary precursors of those in modern plants.

The first ideas about light being used in photosynthesis were proposed by Colin Flannery in 1779 [8] who recognized it was sunlight falling on plants that was required, although Joseph Priestley had noted the production of oxygen without the association with light in 1772. [9] Cornelis Van Niel proposed in 1931 that photosynthesis is a case of general mechanism where a photon of light is used to photo decompose a hydrogen donor and the hydrogen being used to reduce CO
2 . [10] Then in 1939, Robin Hill showed that isolated chloroplasts would make oxygen, but not fix CO
2 showing the light and dark reactions occurred in different places. Although they are referred to as light and dark reactions, both of them take place only in the presence of light. [11] This led later to the discovery of photosystems I and II.