Photosynthesis in Higher Plants

Photosynthesis means basically "to put together with the help of light". This is a series of processes in which electromagnetic energy is converted to chemical energy used for biosynthesis of organic cell materials. Photosynthesis is important due to two reasons: it is the primary source of all food on earth. It is also responsible for the release of oxygen into the atmosphere by green plants.

Green plants carry out ‘photosynthesis’, a physicochemical process by which they use light energy to drive the synthesis of organic compounds.

Photosynthesis is a light-driven oxidation-reduction(Redox) reaction where the energy from the light is used to oxidize water, releasing oxygen gas and hydrogen ions, followed by the transfer of electrons to carbon dioxide, reducing it to organic molecules or carbohydrates.

Photosynthetic organisms are called autotrophs because they can synthesize chemical fuels such as glucose from carbon dioxide and water by utilizing sunlight as an energy source.

Ultimately, all living forms on earth depend on sunlight for energy. The use of energy from sunlight by plants doing photosynthesis is the basis of life on earth. 

1.0Where Does Photosynthesis Take Place? 

Photosynthesis does take place in the green leaves of plants.The mesophyll cells in the leaves, have a large number of chloroplasts. Usually the chloroplasts align themselves along the walls of the mesophyll cells, such that they get the optimum quantity of the incident light.

Within the chloroplast, there is a membranous system consisting of grana, the stroma lamellae, and the matrix stroma. 

There is a clear division of labour within the chloroplast. 

The membrane system is responsible for trapping the light energy and also for the synthesis of ATP and NADPH. A set of reactions, since they are directly light driven are called light reactions (photochemical reactions).

In stroma, enzymatic reactions synthesize sugar, which in turn forms starch. By convention, as dark reactions (carbon reactions). 

2.0Pigments Involved in Photosynthesis 

Pigments are substances that have the ability to absorb light, at specific wavelengths. 

The leaf pigments show that the colour that we see in leaves is not due to a single pigment but due to four pigments: 

Chlorophyll a, the most abundant plant pigment in the world (bright or blue-green in the chromatogram), chlorophyll b (yellow-green), xanthophylls (yellow) and carotenoids (yellow to yellow-orange).

Photosynthetic pigments are organised into two discrete photochemical light-harvesting complexes (LHC) within Photosystem I (PS I) and Photosystem II (PS II). 

The LHC is 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.

The single chlorophyll 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. 

Chlorophyll is a porphyrin derivative conjugated with a magnesium ion that is found in plant chloroplasts, algae and cyanobacteria. Chlorophyll is essential to the process of photosynthesis. It absorbs light in the blue and red parts of the visible spectrum and transfers the absorbed photon energy to an electron, which is used to produce ATP.

There are various types of chlorophyll, the most essential of which is chlorophyll “a”. This is the molecule that enables photosynthesis by sending energised electrons to molecules that produce sugars. 

The second type of chlorophyll is chlorophyll “b”, which is found only in “green algae” and plants. 

A third frequent form of chlorophyll is known as chlorophyll “c”, and it is present solely in photosynthetic members of the Chromista as well as dinoflagellates. 

Synthesis of Chlorophyll

Chlorophyll is produced within the chloroplast from an abundant precursor, glutamate. The processes occur in the plastid stroma and are catalysed by soluble enzymes from glutamate to the tetrapyrrole protoporphyrin IX, where the route splits between chlorophyll and heme.

Accessory pigments, also absorb light and transfer the energy to chlorophyll a. Indeed, they not only enable a wider range of wavelengths of incoming light to be utilised for photosynthesis but also protect chlorophyll from photo-oxidation.

What is a Light Reaction? 

• The initial stage of photosynthesis is the light reaction, in which solar energy is transformed into chemical energy in the form of ATP and NADPH. 
• Protein complexes and pigment molecules both contribute to the synthesis of NADPH and ATP.
• Light reactions or the ‘Photochemical’ phase include light absorption, water splitting, oxygen release, and the formation of high-energy chemical intermediates, ATP and NADPH. 

3.0Electron Transport System and Chemiosmotic Hypothesis

The Electron Transport system (ETS) and Chemiosmotic hypothesis(Photophosphorylation):

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 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. 

Difference Between Cyclic Photophosphorylation and  Non Cyclic Photophosphorylation

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.

Chemiosmosis requires a membrane, a proton pump, a proton gradient and ATP synthase. In photosynthesis too, ATP synthesis is linked to the development of a proton gradient across a membrane. 

The proton accumulation is towards the inside of the membrane, i.e., in the lumen. 

What causes the proton gradient across the membrane? 

(i) Since the 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.

(ii) As electrons move through the photosystems, protons are transported across the membrane. This happens because the primary accepter of electron (PQ) 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. 

(iii) The NADP reductase enzyme is located on the stroma side of the membrane. Along with electrons that come from the acceptor 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 an 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 the synthesis of ATP. The gradient is broken down due to the movement of protons across the membrane to the stroma through the transmembrane channel of the CF0 of the ATP synthase. 

The ATP synthase enzyme consists of two parts: one called the CF0 is embedded in the thylakoid membrane and forms a transmembrane channel that carries out facilitated diffusion of protons across the membrane. 

The other portion is called CF1 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 CF1 particle of the ATP synthase, which makes the enzyme synthesise several molecules of energy packed ATP. 

Energy is used to pump protons across a membrane, to create a gradient or a high concentration of protons within the thylakoid lumen. 

ATP synthase has a channel that allows the diffusion of protons back across the membrane; this releases enough energy to activate the ATP synthase 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 or carbon reaction taking place in the stroma, responsible for fixing CO₂, and synthesis of sugars.

4.0Where are the ATP and NADPH Used?

(The carbon dioxide assimilation reactions of photosynthesis)

The products of the light reactions, NADPH and ATP, are used to synthesize carbohydrates from carbon dioxide, a process called carbon dioxide fixation.

Carbon fixation fundamentally involves the use of the ‘reducing power’ of NADPH to reduce carbon dioxide, and the process is summarized in the following equation:

NADPH + ATP + CO₂ (CH₂ O)n + NADP⁺ + ADP + iP

Note that one of the extremely important aspects of these reactions is that they regenerate metabolites needed in the light reactions: NADP⁺, ADP and iP. 

Since the supplies of these metabolites are limited, it is critical that they be recycled. For ease of understanding, the Calvin cycle can be described under three stages: carboxylation, reduction and regeneration.

1. Carboxylation:

Carboxylation occurs when carbon dioxide is added to a metabolite called ribulose bisphosphate (RuBP), a five-carbon sugar with two phosphates, in a reaction catalyzed by an enzyme called ribulose bisphosphate carboxylase (rubisco). The resultant 6-carbon compound rapidly breaks down into two molecules of a three-carbon compound called phosphoglycerate (PGA).

2. Reduction:

• The PGA is not a very useful compound because it is too oxidized. To be useful the PGA needs to be reduced. It can then be used as a precursor molecule to make a variety of biomolecules such as sugars, amino acids, nucleic acids and many others. 
• In addition, the reduced compound can be used to make more RUBP and thus allow more carbon dioxide to be assimilated.
• The reduction of PGA is accomplished using NADPH and ATP produced in the light reactions of photosynthesis and produces a three-carbon sugar called glyceraldehyde-3-phosphate (G3P).

PGA + NADPH + ATP — > G3P + NADP+ + ADP + iP

3. Regeneration of RuBP:

• In order to sustain photosynthesis the plant needs to regenerate RUBP, the 5-carbon sugar that is used to acquire CO2. This occurs when RUBP is synthesized from G3P. Obviously, you can’t make a five-carbon sugar out of a three-carbon sugar. 
• You might do it using two G3P molecules but there would be one ‘fixed’ carbon leftover. However, the synthesis can be accomplished fifteen if one starts with five G3P molecules (fifteen total carbons) and makes three RUBPs (also fifteen carbons). 

These reactions are called the Calvin-Benson cycle and they require one ATP made in the light reactions for each RUBP produced.

At the same time, G3P can be used to make six-carbon sugars, in particular glucose and fructose and from them, sucrose, starch, cellulose and a wide variety of polysaccharides.

Putting both these activities together, if six molecules of carbon dioxide are fixed by carboxylating six RUBP’s, then 12 G3P can be produced after reduction utilizing 12 NADPH and 12 ATP. 

Ten molecules of G3P can be used to regenerate the six RUBP’s and this process requires six more ATP. The remaining two molecules of G3P can be used to form a fructose or a glucose. This is how all plants carry out photosynthesis. Each carbon dioxide assimilated requires two NADPH and three ATP.

There is a great deal of chemistry taking place in chloroplasts, although the net effect can be expressed simply as

5.0The C4 Pathway

The C4 Pathway (C4 cycle or Hatch and Slack Pathway or Double CO2 Fixation):

Plants that are adapted to dry tropical regions have the C4 pathway. C4 plants tolerate higher temperatures, they show a response to high light intensities, they lack a process called photorespiration and have greater productivity of biomass.

In this cycle, the first formed stable compound is a 4 carbon compound viz., oxaloacetic acid. Hence it is called C₄ cycle. 

The path way is also called as Hatch and Slack as they worked out the pathway in 1966 and it is also called as C₄ dicarboxylic acid pathway. This pathway is commonly seen in many grasses, sugar cane, maize, sorghum and amaranthus. 

The C₄ plants show a different type of leaf anatomy. The chloroplasts are dimorphic in nature. In the leaves of these plants, the vascular bundles are surrounded by bundle sheath of larger parenchymatous cells. 

These bundle sheath cells have chloroplasts. These chloroplasts of bundle sheath are larger, lack grana and contain starch grains. The chloroplasts in mesophyll cells are smaller and always contain grana. 

This peculiar anatomy of leaves of C₄ plants is called Kranz anatomy. The bundle sheath cells are bigger and look like a ring or wreath. 

Kranz in German means wreath and hence it is called Kranz anatomy. The C₄ cycle involves two carboxylation reactions, one taking place in chloroplasts of mesophyll cells and another in chloroplasts of bundle sheath cells. 

There are four steps in Hatch and Slack cycle: 

1. Carboxylation: It takes place in the chloroplasts of mesophyll cells. Phosphoenolpyruvate, a 3-carbon compound picks up CO₂ and changes into 4-carbon oxaloacetate in the presence of water. This reaction is catalysed by the enzyme, phosphoenol pyruvate carboxylase. 

2. Breakdown: Oxaloacetate breaks down readily into 4 carbon malate and aspartate in the presence of the enzyme, transaminase and malate dehydrogenase. These compounds diffuse from the mesophyll cells into sheath cells.

3. Splitting: In the bundle sheath cells, malate and aspartate split enzymatically to yield free CO₂ and 3 carbon pyruvate. The CO₂ is used in Calvin’s cycle in the bundle sheath cell.

The second Carboxylation occurs in the chloroplast of bundle sheath cells. The CO₂ is accepted by 5 carbon compound ribulose diphosphate in the presence of the enzyme, carboxy dismutase and ultimately yields 3 phosphoglyceric acid. Some of the 3 phosphoglyceric acid is utilized in the formation of sugars and the rest regenerate ribulose diphosphate. 

4. Phosphorylation: The pyruvate molecule is transferred to chloroplasts of mesophyll cells where, it is phosphorylated to regenerate phosphoenol pyruvate in the presence of ATP. This reaction is catalysed by pyruvate phosphodikinase and the phophoenol pyruvate is regenerated.

In Hatch and Slack pathway, the C₃ and C₄ cycles of carboxylation are linked and this is due to the Kranz anatomy of the leaves. The C₄ plants are more efficient in photosynthesis than the C₃ plants. The enzyme, phosphoenol pyruvate carboxylase of the C₄ cycle is found to have more affinity for CO₂ than the ribulose diphosphate carboxylase of the C₃ cycle in fixing the molecular CO₂ in organic compound during Carboxylation.

Photorespiration (PCO/Photosynthetic Carbon Oxidation)

An important difference between C₃ and C₄ plants is Photorespiration. The first step of the Calvin pathway – the first CO₂ fixation step. This is the reaction where RuBP combines with CO₂ to form 2 molecules of 3PGA, that is catalysed by RuBisCO. RuBisCO that is the most abundant enzyme in the world is characterised by the fact that its active site can bind to both CO₂ and O₂ – hence the name. 

RuBisCO has a much greater affinity for CO₂ when the CO₂ : O₂ is nearly equal. 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 of phosphoglycerate and phosphoglycolate (2 Carbon) 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.

In C₄ plants photorespiration does not occur. This is because they have a mechanism that increases the concentration of CO₂ at the enzyme site. This takes place when the C₄ acid from the mesophyll is broken down in the bundle sheath 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. 

6.0Factors Affecting Photosynthesis

The rate of photosynthesis is very important in determining the yield of plants including crop plants. Photosynthesis is under the influence of several factors, both internal (plant) and external. 

The plant factors include the number, size, age and orientation of leaves, mesophyll cells and chloroplasts, internal CO₂ concentration and the amount of chlorophyll. The plant or internal factors are dependent on the genetic predisposition and the growth of the plant. 

The external factors would include the availability of sunlight, temperature, CO₂ concentration and water. As a plant photosynthesises, all these factors will simultaneously affect its rate. Hence, though several factors interact and simultaneously affect photosynthesis or CO₂ fixation, usually one factor is the major cause or is the one that limits the rate. Hence, at any point the rate will be determined by the factor available at sub-optimal levels. 

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. For example, despite the presence of a green leaf and optimal light and CO₂ conditions, the plant may not photosynthesise if the temperature is very low. This leaf, if given the optimal temperature, will start photosynthesizing. 

Light :

• We need to distinguish between light quality, light intensity and the duration of exposure to light, while discussing light as a factor that affects photosynthesis. 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. 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 CO₂ conditions. At high light intensities, both C₃ and C₄ plants show increase in the rates of photosynthesis. 
• 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.

Water:

• In the process of photosynthesis, water is a necessary raw ingredient. Photosynthesis consumes less than 1% of the water taken by a plant. Photosynthesis is reduced as the water content of the soil decreases from field capacity to the permanent wilting limit.

Temperature:

• Although the light-dependent reactions of photosynthesis are not affected by changes in temperature, the light independent reactions of photosynthesis are dependent on temperature. They are reactions catalysed by enzymes. As the enzymes approach their optimum temperatures the overall rate increases. It approximately doubles for every 10°C increase in temperature. Above the optimum temperature the rate begins to decrease, as enzymes are denatured, until it stops.