Photosynthesis

Carbon dioxide is converted into sugars in a process called carbon fixation. Carbon fixation is a redox reaction, so photosynthesis needs to supply both a source of energy to drive this process, and also the electrons needed to convert carbon dioxide into carbohydrate, which is a reduction reaction. In general outline, photosynthesis is the opposite of cellular respiration, where glucose and other compounds are oxidized to produce carbon dioxide, water, and release chemical energy. However, the two processes take place through a different sequence of chemical reactions and in different cellular compartments.

The general equation for photosynthesis is therefore:

2n CO₂ + 2n H₂A + photons → 2(CH₂O)n + n O₂ + 2n A

carbon dioxide + electron donor + light energy → carbohydrate + oxygen + oxidized electron donor

Since water is used as the electron donor in oxygenic photosynthesis, the equation for this process is:

2n CO₂ + 2n H₂O + photons → 2(CH₂O)n + 2n O₂

carbon dioxide + water + light energy → carbohydrate + oxygen

Other processes (e.g. as used by microbial species in Mono Lake, California) substitute other compounds (such as arsenite) for water in the electron-supply role; the microbes use sunlight to oxidize arsenite to arsenate. The equation for this reaction is:

(AsO₃^3-) + CO₂ + photons → CO + (AsO₄^3-)

carbon dioxide + arsenite + light energy → arsenate + carbon monoxide (used to build other compounds in subsequent reactions)

Photosynthesis occurs in two stages. In the first stage, light-dependent reactions or light reactions capture the energy of light and use it to make the energy-storage molecules ATP and NADPH. During the second stage, the light-independent reactions use these products to capture and reduce carbon dioxide.

Most organisms that utilize photosynthesis to produce oxygen use visible light, at least three organisms use infrared radiation.

Photosynthetic Membranes And Orgenelles

In plants and algae, photosynthesis takes place in organelles called chloroplasts. A typical plant cell contains about 10 to 100 chloroplasts. The chloroplast is enclosed by a membrane. This membrane is composed of a phospholipid inner membrane, a phospholipid outer membrane, and an intermembrane space between them. Within the membrane is an aqueous fluid called the stroma. The stroma contains stacks (grana) of thylakoids, which are the site of photosynthesis. The thylakoids are flattened disks, bounded by a membrane with a lumen or thylakoid space within it. The site of photosynthesis is the thylakoid membrane, which contains integral and peripheral membrane protein complexes, including the pigments that absorb light energy, which form the photosystems.

Plants absorb light primarily using the pigment chlorophyll, which is the reason that most plants have a green color. Besides chlorophyll, plants also use pigments such as carotenes and xanthophylls. Algae also use chlorophyll, but various other pigments are present as phycocyanin, carotenes, and xanthophylls in green algae, phycoerythrin in red algae (rhodophytes) and fucoxanthol in brown algae and diatoms resulting in a wide variety of colors.

These pigments are embedded in plants and algae in special antenna-proteins. In such proteins all the pigments are ordered to work well together. Such a protein is also called a light-harvesting complex.

Although all cells in the green parts of a plant have chloroplasts, most of the energy is captured in the leaves. The cells in the interior tissues of a leaf, called the mesophyll, can contain between 450,000 and 800,000 chloroplasts for every square millimeter of leaf. The surface of the leaf is uniformly coated with a water-resistant waxy cuticle that protects the leaf from excessive evaporation of water and decreases the absorption of ultraviolet or blue light to reduce heating. The transparent epidermis layer allows light to pass through to the palisade mesophyll cells where most of the photosynthesis takes place.

(ref: http://en.wikipedia.org/wiki/File:Chloroplast.svg accessed on 10th Oct, 2009)

LIGHT REACTION

Light reactions or the ‘Photochemical’ phase include light absorption, water splitting, oxygen release, and the formation of high-energy chemical intermediates, ATP and NADPH. Several complexes are involved in the process. The pigments are organised into two discrete photochemical light harvesting complexes (LHC) within the Photosystem I (PS I) and Photosystem II (PS II). These are named in the sequence of their discovery, and not in the sequence in which they function during the light reaction. The LHC are made up of hundreds of pigment molecules bound to proteins. Each photosystem has all the pigments (except one molecule of chlorophyll a) forming a light harvesting system also called antennae. These pigments help to make photosynthesis more efficient by absorbing different wavelengths of light. The single chlorophyll a molecule forms the reaction centre. The reaction centre is different in both the photosystems. In PS I the reaction centre chlorophyll a has an absorption peak at 700 nm, hence is called P700, while in PS II it has absorption maxima at 680 nm, and is called P680.

THE ELECTRON TRANSPORT

In photosystem II the reaction centre chlorophyll a absorbs 680 nm wavelength of red light causing electrons to become excited and jump into an orbit farther from the atomic nucleus. These electrons are picked up by an electron acceptor which passes them to an electrons transport system consisting of cytochromes.

This movement of electrons is downhill, in terms of an oxidation-reduction or redox potential scale. The electrons are not used up as they pass through the electron transport chain, but are passed on to the pigments of photosystem PS I. Simultaneously, electrons in the reaction centre of PS I are also excited when they receive red light of wavelength 700 nm and are transferred to another accepter molecule that has a greater redox potential.

Z Scheme: These electrons then are moved downhill again, this time to a molecule of energy-rich NADP+. The addition of these electrons reduces NADP⁺ to NADPH+H⁺. This whole scheme of transfer of electrons, starting from the PS II, uphill to the accepter, down the electron transport chain to PS I, excitation of electrons, transfer to another accepter, and finally down hill to NADP⁺ causing it to be reduced to NADPH+H⁺ is called the Z scheme, due to its characterstic shape. This shape is formed when all the carriers are placed in a sequence on a redox potential scale.

Splitting of Water

The splitting of water is associated with the PS II; water is split into H+, [O] and electrons. This creates oxygen, one of the net products of photosynthesis. The electrons needed to replace those removed from photosystem I are provided by photosystem II.

2H₂O → 4H⁺ + O₂ + 4^e-

Cyclic and Non-cyclic Photo-phosphorylation

Living organisms have the capability of extracting energy from oxidisable substances and store this in the form of bond energy. Special substances like ATP, carry this energy in their chemical bonds. The process of which ATP is synthesised by cells (in mitochondria and chloroplasts) is named phosphorylation. Photophosphorylation is the synthesis of ATP from ADP and inorganic phosphate in the presence of light.

When the two photosystems work in a series, first PS II and then the PS I, a process called non-cyclic photo-phosphorylation occurs.

The two photosystems are connected through an electron transport chain, as seen earlier – in the Z scheme. Both ATP and NADPH+H⁺ are synthesised by this kind of electron flow.

When only PS I is functional, the electron is circulated within the photosystem and the phosphorylation occurs due to cyclic flow of electrons. A possible location where this could be happening is in the stroma lamellae. While the membrane or lamellae of the grana have both PS I and PS II the stroma lamellae membranes lack PS II as well as NADP reductase enzyme. The excited electron does not pass on to NADP⁺ but is cycled back to the PS I complex through the electron transport chain. The cyclic flow hence, results only in the synthesis of ATP, but not of NADPH+H⁺. Cyclic photophosphorylation also occurs when only light of wavelengths beyond 680 nm are available for excitation.

Chemiosmotic Hypothesis

Like in respiration, in photosynthesis too, ATP synthesis is linked to development of a proton gradient across a membrane. This time these are membranes of the thylakoid. There is one difference though, here the proton accumulation is towards the inside of the membrane, i.e., in the lumen. In respiration, protons accumulate in the intermembrane space of the mitochondria when electrons move through the ETS. Let us understand what causes the proton gradient across the membrane. We need to consider again the processes that take place during the activation of electrons and their transport to determine the steps that cause a proton gradient to develop.

(a) Since splitting of the water molecule takes place on the inner side of the membrane, the protons or hydrogen ions that are produced by the splitting of water accumulate within the lumen of the thylakoids.

(b) As electrons move through the photosystems, protons are transported across the membrane. This happens because the primary accepter of electron which is located towards the outer side of the membrane transfers its electron not to an electron carrier but to an H carrier. Hence, this molecule removes a proton from the stroma while transporting an electron. When this molecule passes on its electron to the electron carrier on the inner side of the membrane, the proton is released into the inner side or the lumen side of the membrane.

(c) The NADP reductase enzyme is located on the stroma side of the membrane. Along with electrons that come from the accepter of electrons of PS I, protons are necessary for the reduction of NADP⁺ to NADPH+H⁺. These protons are also removed from the stroma.

Hence, within the chloroplast, protons in the stroma decrease in number, while in the lumen there is accumulation of protons. This creates a proton gradient across the thylakoid membrane as well as a measurable decrease in pH in the lumen.

This gradient is important because it is the breakdown of this gradient that leads to release of energy. The gradient is broken down due to the movement of protons across the membrane to the stroma through the transmembrane channel of the F₀ of the ATPase. The ATPase enzyme consists of two parts: one called the F0 is embedded in the membrane and forms a transmembrane channel that carries out facilitated diffusion of protons across the membrane. The other portion is called F1 and protrudes on the outer surface of the thylakoid membrane on the side that faces the stroma. The break down of the gradient provides enough energy to cause a conformational change in the F1 particle of the ATPase, which makes the enzyme synthesise several molecules of energy-packed ATP.

Chemiosmosis requires a membrane, a proton pump, a proton gradient and ATPase. Energy is used to pump protons across a membrane, to create a gradient or a high concentration of protons within the thylakoid lumen. ATPase has a channel that allows diffusion of protons back across the membrane; this releases enough energy to activate ATPase enzyme that catalyses the formation of ATP. Along with the NADPH produced by the movement of electrons, the ATP will be used immediately in the biosynthetic reaction taking place in the stroma, responsible for fixing CO₂, and synthesis of sugars.

ATP AND NADPH

Products of light reaction are ATP, NADPH and O₂. Of these O₂ diffuses out of the chloroplast while ATP and NADPH are used to drive the processes leading to the synthesis of food, more accurately, sugars. This is the biosynthetic phase of photosynthesis. This process does not directly depend on the presence of light but is dependent on the products of the light reaction, i.e., ATP and NADPH, besides CO₂ and H₂O.

The Calvin Cycle

Calvin and his co-workers then worked out the whole pathway and showed that the pathway operated in a cyclic manner; the RuBP (Ribulose bisphosphate) was regenerated.

The Calvin cycle can be described under three stages: carboxylation, reduction and regeneration.

1. Carboxylation – Carboxylation is the fixation of CO₂ into a stable organic intermediate. Carboxylation is the most crucial step of the Calvin cycle where CO₂ is utilised for the carboxylation of RuBP. This reaction is catalysed by the enzyme RuBP carboxylase which results in the formation of two molecules of 3-PGA. Since this enzyme also has an oxygenation activity it would be more correct to call it RuBP carboxylase-oxygenase or RuBisCO.

2. Reduction – These are a series of reactions that lead to the formation of glucose. The steps involve utilisation of 2 molecules of ATP for phosphorylation and two of NADPH for reduction per CO₂ molecule fixed. The fixation of six molecules of CO₂ and 6 turns of the cycle are required for the removal of one molecule of glucose from the pathway.

3. Regeneration – Regeneration of the CO₂ acceptor molecule RuBP is crucial if the cycle is to continue uninterrupted. The regeneration steps require one ATP for phosphorylation to form RuBP.

Hence for every CO₂ molecule entering the Calvin cycle, 3 molecules of ATP and 2 of NADPH are required. It is probably to meet this difference in number of ATP and NADPH used in the dark reaction that the cyclic phosphorylation takes place.

To make one molecule of glucose 6 turns of the cycle are required.

(ref: http://en.wikipedia.org/wiki/File:Calvin-cycle3.png accessed on 10th Oct 2009)

THE C4 PATHWAY

Plants that are adapted to dry tropical regions have the C4 pathway. Though these plants have the C4 oxaloacetic acid as the first CO₂ fixation product they use the C3 pathway or the Calvin cycle as the main biosynthetic pathway.

C4 plants are special: They have a special type of leaf anatomy, they tolerate higher temperatures, they show a response to highlight intensities, they lack a process called photorespiration and have greater productivity of biomass. Let us understand these one by one.

(Ref: http://en.wikipedia.org/wiki/File:HatchSlackpathway.png accessed on 10th Oct, 2009)

The particularly large cells around the vascular bundles of the C4 pathway plants are called bundle sheath cells, and the leaves which have such anatomy are said to have ‘Kranz’ anatomy. ‘Kranz’ means ‘wreath’ and is a reflection of the arrangement of cells. The bundle sheath cells may form several layers around the vascular bundles; they are characterised by having a large number of chloroplasts, thick walls impervious to gaseous exchange and no intercellular spaces.

This pathway that has been named the Hatch and Slack Pathway, is again a cyclic process. Let us study the pathway by listing the steps.

The primary CO₂ acceptor is a 3-carbon molecule phosphoenol pyruvate (PEP) and is present in the mesophyll cells. The enzyme responsible for this fixation is PEP carboxylase or PEPcase. It is important to register that the mesophyll cells lack RuBisCO enzyme. The C4 acid OAA is formed in the mesophyll cells.

It then forms other 4-carbon compounds like malic acid or aspartic acid in the mesophyll cells itself, which are transported to the bundle sheath cells. In the bundle sheath cells these C4 acids are broken down to release CO₂ and a 3-carbon molecule.

The 3-carbon molecule is transported back to the mesophyll where it is converted to PEP again, thus, completing the cycle.

The CO₂ released in the bundle sheath cells enters the C3 or the Calvin pathway, a pathway common to all plants. The bundle sheath cells are rich in an enzyme Ribulose bisphosphate carboxylase-oxygenase (RuBisCO), but lack PEPcase. Thus, the basic pathway that results in the formation of the sugars, the Calvin pathway, is common to the C3 and C4 plants.

PHOTORESPIRATION

RuBisCO, that is the most abundant enzyme in the world, is characterised by the fact that its active site can bind to both CO₂and O₂^– hence the name. RuBisCO has a much greater affinity for CO₂ than for O₂. This binding is competitive. It is the relative concentration of O₂ and CO₂ that determines which of the two will bind to the enzyme.

In C3 plants some O₂ does bind to RuBisCO, and hence CO₂ fixation is decreased. Here the RuBP instead of being converted to 2 molecules of PGA binds with O₂ to form one molecule and phosphoglycolate in a pathway called photorespiration. In the photorespiratory pathway, there is neither synthesis of sugars, nor of ATP. Rather it results in the release of CO₂ with the utilisation of ATP. In the photorespiratory pathway there is no synthesis of ATP or NADPH. Therefore, photorespiration is a wasteful process.

In C4 plants photorespiration does not occur. This is because they have a mechanism that increases the concentration of CO₂at the enzyme site. This takes place when the C4 acid from the mesophyll is broken down in the bundle cells to release CO₂^–this results in increasing the intracellular concentration of CO₂. In turn, this ensures that the RuBisCO functions as a carboxylase minimising the oxygenase activity.

FACTORS AFFECTING PHOTOSYNTHESIS

When several factors affect any [bio] chemical process, Blackman’s (1905) Law of Limiting Factors comes into effect. This states the following:

“If a chemical process is affected by more than one factor, then its rate will be determined by the factor which is nearest to its minimal value: it is the factor which directly affects the process if its quantity is changed.”

Light

There is a linear relationship between incident light and CO₂ fixation rates at low light intensities. At higher light intensities, gradually the rate does not show further increase as other factors become limiting. What is interesting to note is that light saturation occurs at 10 per cent of the full sunlight. Hence, except for plants in shade or in dense forests, light is rarely a limiting factor in nature. Increase in incident light beyond a point causes the breakdown of chlorophyll and a decrease in photosynthesis.

Carbon dioxide Concentration

Carbon dioxide is the major limiting factor for photosynthesis. The concentration of CO₂ is very low in the atmosphere (between 0.03 and 0.04 per cent). Increase in concentration upto 0.05 per cent can cause an increase in CO₂ fixation rates; beyond this the levels can become damaging over longer periods.

The C3 and C4 plants respond differently to CO₂ concentrations. At low light conditions neither group responds to high CO₂conditions. At high light intensities, both C3 and C4 plants show increase in the rates of photosynthesis. What is important to note is that the C4 plants show saturation at about 360 μlL^-1 while C3 responds to increased CO₂ concentration and saturation is seen only beyond 450 μlL^-1. Thus, current availability of CO₂ levels is limiting to the C3 plants. The fact that C3 plants respond to higher CO₂ concentration by showing increased rates of photosynthesis leading to higher productivity has been used for some greenhouse crops such as tomatoes and bell pepper. They are allowed to grow in carbon dioxide enriched atmosphere that leads to higher yields.

Temperature

The dark reactions being enzymatic are temperature controlled. Though the light reactions are also temperature sensitive they are affected to a much lesser extent. The C4 plants respond to higher temperatures and show higher rate of photosynthesis while C3 plants have a much lower temperature optimum.

The temperature optimum for photosynthesis of different plants also depends on the habitat that they are adapted to. Tropical plants have a higher temperature optimum than the plants adapted to temperate climates.

Water

Even though water is one of the reactants in the light reaction, the effect of water as a factor is more through its effect on the plant, rather than directly on photosynthesis. Water stress causes the stomata to close hence reducing the CO₂ availability. Besides, water stress also makes leaves wilt, thus, reducing the surface area of the leaves and their metabolic activity as well.