photosynthesis in higher plants

• The green plants make or rather synthesise the food they need through photosynthesis and are therefore called autotrophs.
• Green plants carry out 'photosynthesis', a physico-chemical process by which they use light energy to drive the synthesis of organic compounds.
• 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

• It is the primary source of all food on earth
• It is responsible for the release of oxygen into the atmosphere by green plants.

• Chlorophyll (green pigment of the leaf), light and co2 are required for photosynthesis to occur.

Experiment 1

• A variegated leaf or a leaf that was partially covered with black paper and exposed to light.
• On testing these leaves for the presence of starch, it was clear that photosynthesis occurred only in the green parts of the leaves in the presence of light. Experiment II
Half leaf experiment
• A part of a leaf is enclosed in a test tube containing some KOH soaked cotton (which absorbs co2), while the other half is exposed to air.
• The setup is then placed in light for some time.
• On testing for the presence of starch later in the two parts of the leaf showed that;
Exposed part of the leaf tested positive for starch while the portion that was in the tube, tested negative. This showed that co2was required for photosynthesis.

• Joseph Priestley (1733 - 1804) in 1770 performed a series of experiments that revealed the essential role of air in the growth of green plants
• He discovered oxygen in 1774
• He observed that a candle burning in a closed space - a bell jar, soon gets extinguished
• Similarly, a mouse would soon suffocate in a closed space
• He concluded that a burning candle or an animal that breathe the air, both somehow, damage the air
• He placed a mint plant in the same bell jar
• He found that the mouse stayed alive and the candle continued to burn
• Priestley hypothesised as follows: Plants restore the air whatever breathing animals and burning candles remove

• Jan Ingenhousz (1730 - 1799) conducted the same experiment by placing it once in the dark and once in the sunlight.
• He showed that sunlight is essential to the plant for purifying the air fouled by burning candles or breathing animals.
• He repeated this elegant experiment with an aquatic plant.
• It showed that in bright sunlight, small bubbles were formed around green parts while in the dark they did not.
• Later he identified these bubbles to be of oxygen. Hence he showed that it is only the green part of the plants that could release oxygen.

• In 1854, Julius von Sachs provided evidence for production of glucose when plants grow.
• Chlorophyll is present in special bodies (chloroplast) within plant cell.
• Glucose is made in the green parts of the plants.
• Glucose is usually stored as starch.

• T.W. Engelmann (1843 – 1909) split light using a prism into its spectral components.
• Then illuminated a green alga, (Cladophora), placed in a suspension of aerobic bacteria.
• The bacteria were used to detect the sites of \(O_{2}\) evolution.
• He observed that the bacteria accumulated mainly in the region of blue and red light of the split spectrum.
• A first action spectrum of photosynthesis was thus described.
• It resembles roughly the absorption spectra of chlorophyll a and b.

• By the middle of 19th century, it is discovered that plants use light energy to make carbohydrates from CO₂ and H₂O
• The empirical equation of photosynthesis:
CO2+H2O(in presence of light)--->[CH2O]+O2
• Where[CH2O]represented a carbohydrate (Eg: glucose, a six-carbon sugar).

• Cornelius van Niel (1897-1985) (microbiologist) conducted some studies in purple and green bacteria
• He demonstrated that photosynthesis is essentially a light-dependent reaction, in which hydrogen from a suitable oxidisable compound reduces carbon dioxide to carbohydrates
• This can be represented as;
2H2A+CO2(in presence of light) --->2A+CH2O+H2O
• In green plants H2O is the hydrogen donor and is oxidised to O2
• Purple and green sulphur bacteria use H2S as H donor so 'oxidation' product is sulphur or sulphate depending on the organism and not O2
• Hence, he inferred that the O2 evolved by the green plant comes from H2O, not from carbon dioxide. This was later proved by using radio isotopic techniques •Hence overall correct equation is : 6CO2+12H2O(in presence of light)--->C6H12O6 +6H20+6O2

Chloroplast
• Photosynthesis takes place in green leaves and other green parts of the plants
• Usually chloroplasts align themselves along the walls of mesophyll cells of leaves.
• It helps to get optimum quantity of incident light
• Within the chloroplasts there is a membranous system consisting of grana, stroma lamellae and matrix stroma
• There is a clear division of labour within the chloroplast
• Each granum is a group of membrane bound sacs called thylakoids (lamellae). They contain leaf pigments
• The membrane system traps light energy and synthesis ATP and NADPH. It is called light reaction (photochemical reactions)
• In stroma enzymatic reactions synthesise sugar, which in turn forms starch, it is called dark reaction (carbon reactions)
• Because it is not directly light driven but are dependent on the products of light reactions (ATP & NADPH)

• Pigments are substances that have an ability to absorb light, at specific wavelengths
• Chromatography shows the following pigments in the chromatogram:
• Chlorophyll a (bright or blue green)
• Chlorophyll b (yellow green)
• Xanthophylls (yellow)
• Carotenoids (yellow to yellow – orange). •most of photosynthesis happen in blue and red region. • The absorption spectrum & action spectrum coincide closely showing that; maximum absorption is by chlorophyll a ie, in the blue and red regions, also shows higher rate of photosynthesis
• The graph also shows that chlorophyll a is the chief pigment associated with photosynthesis
• Chlorophyll is the major pigment responsible for trapping light
• Chlorophyll b, xanthophylls and carotenoids are called accessory pigments

• They absorb light at different wavelength and transfer the energy to chlorophyll a
• They protect chlorophyll a from photo-oxidation
Photosynthetically active radiation (PAR) = 400 to 700 nm

• Light reaction (Photochemical phase) includes:
• Light absorption
• Water splitting
• Oxygen release
• Formation of ATP and NADPH (high-energy chemical intermediates)
• 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 discovery & not in the sequence in which they function during light reaction
• LHC are made up of hundreds of pigment molecules bound to proteins
• Each photosystem has all the pigments (except one molecules of chlorophyll a) forming a light harvesting system also called antennae. • They can absorb different wavelengths of light
• The single chlorophyll a molecule forms the reaction centre
• In PS I the reaction centre chlorophyll a has an absorption peak at 700 nm, hence is called P700
• In PS II it has absorption maxima at 680 nm, and is called P680

• When PS II the reaction centre chlorophyll a absorbs red light of 680 nm wavelength, electrons are excited and transferred to an electron acceptor.
• The electron acceptor passes them to a chain of electron transport system consisting of cytochromes.
• This movement of electrons is downhill in terms of an oxidation reduction or redox potential scale.
• The electrons are transferred to the pigments of 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 acceptor molecule having a great redox potential.
• These electrons then are moved downhill to a molecule of energy-rich NADP^+. As a result NADP+ reduced to NADPH+H^+
• This whole scheme of transfer of electrons, starting from the PS II, uphill to the acceptor, down the ETC to PS I, excitation of electrons, transfer to another acceptor, and finally down

• The water splitting complex in PS II is physically located on the inner side of the thylakoid membrane
• Water is split into 2H+,[O]and electrons
2H2O--->4H+ + O2+4electron
• This creates oxygen, one of the net products of photosynthesis.
• So, PS II can supply electrons continuously by replacing electrons from water splitting
• Thus PS II provides electrons needed to replace those removed from PS I
• The protons H+ are used to reduce NADP+ to NADPH

• Living organisms have the capability of extracting energy from oxidisable substances and store as bond energy
• ATP, carry this energy in their chemical bonds
• The synthesis of ATP by cells (in mitochondria and chloroplasts) is called phosphorylation
• Photophosphorylation is the synthesis of ATP from ADP and inorganic phosphate in the presence of light

• It occurs when the two photosystems work in a series (first PS II and then PS I)
• The two photosystems are connected through an electron transport chain as seen earlier in the z-scheme
• Here, ATP and NADPH + H+ are synthesised
• It is a non-cyclic process because the electrons lost by PS II do not come back to it but pass on to NADP+
Splitting of H20: 2H20 ---> 4H+ +.O2+4electron

• It occurs in stroma lamellae when only PS I is functional
• The electron is circulated within the photosystem and the phosphorylation occurs due to cyclic flow of electrons
• 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
• Cyclic flow hence, results only in the synthesis of ATP (no NADPH + H⁺).
• Cyclic photophosphorylation also occurs when only light of wavelengths beyond 680 nm are available for excitation

• It explains the mechanism of ATP synthesised in chloroplast.
• Like in respiration, in photosynthesis too, ATP synthesis is linked to development of a proton gradient across a membrane of thylakoid.
• Here proton accumulation is towards the inside of the membrane ie, in lumen.
• In respiration proton accumulate in the inter membrane space of mitochondria, when electron move through the ETS.
STEPS
a) Splitting of the water molecule takes place on the inner side of the membrane. So the protons accumulate in the lumen of thylakoids

b) As electrons move through the photosystems, protons are transported across the membrane:

· Because the primary acceptor 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
· 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 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 are decrease in number, but in lumen there is an accumulation of protons.

This creates a proton gradient across the thylakoid membrane and decrease in pH in the lumen.

Breakdown of proton gradient leads to synthesis of ATP by ATP synthase enzyme.

The gradient is broken down due to the movement of protons across the membrane to the stroma through transmembrane channel of CF₀ of ATP synthase. Chemiosmosis requires a membrane, a proton pump, a proton gradient and ATP synthase
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 diffusion of protons back across the membrane
This releases enough energy to activate 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 taking place in the stroma
It is responsible for fixing CO₂, and synthesis of sugars.

CF₀: It is embedded in the thylakoid membrane and forms a trans-membrane channel that carries out facilitated diffusion of protons across the membrane to the stroma. It results in breakdown of proton gradient CF₁: It protrudes on the outer surface of the thylakoid membrane. The energy due to breakdown of gradient cause a conformational change in the CF₁ particle. It makes the enzymes to synthesise ATP molecule.

The products of light reaction are ATP, NADPH and O₂
· Biosynthetic phase (Dark reaction) is the use of ATP and NADPH to drive the process for the synthesis of food (sugar).
· This process does not directly depend on the presence of light but is dependent on the products of the light reaction.It can be verified as follows:
immediately after light becomes unavailable, the biosynthetic process continues for some time, and then stops. If then, light is made available, the synthesis starts again
· CO₂ is combined with H₂O to produce (CH₂O)n or sugars.
· CO₂ assimilation during photosynthesis is two types:

C₃ pathway: In this, first stable product of CO₂ fixation is a C₃ organic acid-3-phosphoglyceric acid (PGA). Melvin Calvin discovered this using ¹⁴C in algal photosynthesis.
C₄ pathway: In this first stable product is oxaloacetic acid (OAA), a C₄ acid.

The acceptor molecule was a 5-carbon ketose sugar – ribulose bisphosphate (RuBP).
· Scientists believed that, since the first product was a C₃ acid, the primary acceptor would be a 2-carbon compound.
· They spent many years trying to identify a 2-carbon compound before they discovered the 5-carbon RuBP.

Calvin and his co-workers showed that the pathway operated in a cyclic manner.
· The RuBP was regenerated
· The Calvin pathway occurs in all photosynthetic plants
· It does not matter whether they have C₃ or C₄ (or any other) pathways.

IT HAS THREE STAGES

1. CARBOXYLATION
2. REDUCTION
3. REGENERATION

· It is the fixation of CO₂ into a stable organic intermediate
· It 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 is called RuBP carboxylase-oxygenase or RuBisCO

· 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
· Fixation of 6CO₂ molecules and 6 turns of the cycle are required for the formation of one molecule of glucose from the pathway

· Regeneration of the RuBP is crucial if the cycle is to continue uninterrupted
· It require one ATP for phosphorylation to form RuBP
· Hence for every CO₂ molecule, 3 ATP molecules and 2 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.

In | Out
Six CO₂ | One glucose
18 ATP | 18 ADP
12 NADPH | 12 NADP

· It is present in plants adapted to dry tropical regions
· They also 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 temperature
· They show a response to high light intensities
· They lack a process called photorespiration
· They have greater productivity of biomass · The large cells around the vascular bundles of the C₄ plants are called bundle sheath cells. Such anatomy is called 'Kranz' anatomy. ('Kranz' means 'wreath').
· The bundle sheath cells may form several layers around the vascular bundles. · They have large number of chloroplasts, thick walls impervious to gaseous exchange and no intercellular spaces. (Handwritten note above "intercellular spaces": chloroplasts)
· Presence of bundle sheath helps us to identify C₄ plants.
· Examples for C₄ plants are Maize, Sugarcane and Sorghum · 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.
· 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 cell. They 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. (Pyruvate)
· The 3C 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 pathway or calvin pathway.
· The bundle sheath cells are rich in RuBisCO, but lack PEPcase. Thus, the basic pathway results in formation of sugar the Calvin pathway is common to the C₃ and C₄ plants.

· In Calvin pathway RuBP combines with CO₂ to form 2 molecules of 3 PGA
RuBP + CO₂ (in presence of RuBisco) gives 2×3PGA
· This reaction is catalysed by RuBisCO
· RuBisCO is the most abundant enzyme in the world.
· 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.
· The binding is competitive. It is the relative-concentration of O₂ and CO₂ that determines which of the two will bind to the enzyme.· Here the RuBP binds with O₂ to form one molecule of phosphoglycerate and phosphoglycolate in a pathway called photorespiration. also called C2 cycle. · In C₃ plants some O₂ does bind to RuBisCO, and hence CO₂ fixation is decreased
· In this pathway, there is no synthesis of sugars, ATP and NADPH
· Hence photorespiration is a wasteful process. Rather it causes the release of CO₂ by using ATP.
· 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 minimises the oxygenase activity of RuBisCO
· Due to the lack of photorespiration, productivity and yields are better in C₄ plants.

Internal (plant) factors

· Number, size, age and orientation of leaves, mesophyll cells & chloroplast.
· Internal CO₂ concentration.
· Amount of chlorophyll.
· The plant or internal factors are dependent on the genetic predisposition and growth of the plant.

External factors

· Sunlight
· Temperature
· CO₂ concentration
· Water

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

· Eg: A plant with green leaf, optimal light & CO₂ may not photosynthesise in very low temperature. If optimal temperature is given, it will start photosynthesising

· Light quality, light intensity and the duration of exposure to light influence photosynthesis.
· There is a linear relationship between incident light and CO₂ fixation 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.
· High increase in incident light break down chlorophyll. It decreases photosynthesis.

· It is the major limiting factor for photosynthesis.
· CO₂ concentration is very low in the atmosphere (0.03 - 0.04%). · Increase up to 0.05% can cause an increase in CO₂ fixation rates; beyond this the levels can become damaging over longer periods.
· At low light conditions C₃ and C₄ plants do not respond to high CO₂ conditions. At high light intensities, they show increased rate of photosynthesis.
· C₄ plants show saturation at about 360 µL L⁻¹
· C₃ responds to increased CO₂ concentration and saturation is seen only beyond 450 µL L⁻¹
· Thus, current availability of CO₂ levels is limiting to the C₃ plants.
· At higher CO₂ concentration, C₃ plants shows increased photosynthesis and higher productivity.
· This fact is used for some greenhouse crops (tomatoes and bell pepper).

· The dark reactions being enzymatic are temperature controlled
· Influence of temperature on light reaction is very less
· The C₄ plants respond to higher temperatures and show higher rate of photosynthesis
· C₃ plants have a much lower temperature optimum
· The temperature optimum of plants also depends on their habitat
· Tropical plants have a higher temperature optimum than the plants adapted to temperate climates

· Water stress causes the stomata to close hence reducing the CO₂ availability
· Water stress also makes leaves wilt, thus, reducing the surface area of the leaves and their metabolic activity as well