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 CO₂ conditions. 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 μL^-1while C₃ responds to increased CO₂ concentration and saturation is seen only beyond 450 μ1L^-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.

Steps of C₄ pathway:
(i) The primary CO₂ acceptor is a 3-carbon molecule phosphoenol pyruvate (PEP) and is present in the mesophyll cells. The enzyme responsible for this flxation is PEP carboxylase or PEP case. It is important to register that the mesophyll cells lack RuBisCO enzyme. The C₄ acid OAA is formed in the mesophyll cells.
(ii) 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.

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

The Calvin Cycle
Calvin and his co-workers 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.
The Calvin Cycle
Importance of Calvin Cycle. Calvin cycle is the major step in which carbon fixation takes place. In other words this can be said as the 'moment of truth' in photosynthesis.

The diagram is showing cyclic photophosphorylation.Here, A is electron acceptor, B is electron transport system and C is Photo system-I\({ PS }_{ 400 }\)
In cyclic photophosphorylation, only PS-I is functional.The electron is circulated within the photosystem and the phosphorylation occurs due to cyclic flow of electrons.

(i) It is the relative rates of photosynthesis at different wavelengths of light.
(ii) Absorption of different wavelengths of light by a particular pigment is plotted and is called the absorption spectrum of that pigment. 
(iii) Chlorophyll-a is responsible for light reaction of photosynthesis, but the action spectrum and absorption spectrum do not overlap because, though chlorophyll is the main pigment responsible for absorption of light, other thylakoid pigments like chlorophyll-b, xanthophylls and carotenoids, which are called accessory pigments. also absorb and transfer the energy to chlorophyll-a. Indeed they not only enable a wider range of wavelength of incoming light to be utilised for photosynthesis, but also protect chlorophyll-a from photooxidation.

C₄-plants can produce more suger than C₃-plants in condition of bright light and high tempertaure.It is because of the following reasons.
i)They have a special type of leaf anatomy
ii)They can tolerate higher temperature.
iii)They show response to high light intensities
iv)They lack a process called photorespiration.
v)They have greater productivity of biomass.
Many important crop plants are C₄-plants including maize, sorghum, sugarcane and millet.

RuBisCO has a much greater affinity for CO₂ than for O₂ under normal conditions.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 utilization of ATP. In the photorespiratory pathway, there is no synthesis of ATP or NADPH. Therefore, photorespiration is a wasteful process.
In C,•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 C, acid frorn the mesophyll is brcoe down in the bundle cells to release CO₂this increasing the intracellular concentration ofCO₂ turn, thís ensures that the RuBisCO functions carboxylase minimising the oxygenase activity

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 characterized by having a large number of chloroplasts, thick walls impervious to gaseous exchange and no intercellular spaces.
C₄ -plants can produce more sugar than C₃-plants in conditions of bright light and high temperature.Many important crop plants are C₄-plants, i.e maize, sorghum, sugarcane, and millet.

Although all cells in the green part 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 450000 and 800000 chloroplasts for every square millimetre of leaf (nearly 60-70 chloroplasts/cell).
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 epidermal layer allows light to pass through to the palisade mesophyll cells, where most of the photosynthesis takes place.The green stems are also capable of performing photosynthesis.

Comparison between the cyclic and non-cyclic photophosphorylation