photosynthesis

chloroplast

• chloroplasts are organelles that contain photosynthetic pigments, enzymes, and other intermediates to carry out photosynthesis

  • contain circular DNA & 70S ribosomes

  • vary greatly in numbers and shapes from one type of plant to another

  • typically found in palisade mesophyll cells and spongy mesophyll cells in leaves

  • root hair cells do not have chloroplasts, as these cells do not carry out photosynthesis

structure of chloroplast

1. disc-shaped

2. bounded by a double membrane → envelope

   • both outer and inner membrane form smooth continuous boundaries

   • inner membrane is impermeable to ions

     • during chloroplast development, the inner membrane project inwards to give rise to fluid filled sacs called thylakoids but it becomes disconnected from thylakoids in mature chloroplasts

3. thylakoids → third membrane system

   • thylakoid membrane forms a network of flattened discs known as thylakoids

     • thylakoids are usually stacked to form grana (singular: granum)

     • grana can be joined by intergranal lamella (plural: lamellae)

   • thylakoid membrane contains the following which are embedded or attached on its surface

     1. photosystems containing various light-absorbing photosynthetic pigments (e.g. chlorophyll, carotenoids) for absorption of light energy during light reaction of photosynthesis

     2. electron carriers

     3. enzymes (e.g., NADP+ reductase)

     4. stalked particles containing ATP synthase

   • extensive thylakoid membrane increases surface area for absorption of light energy

   • inside the thylakoid is the thylakoid space → important to create a steep proton gradient for chemiosmosis

4. stroma (fluid-filled space) contains:

   • circular DNA with genes which code for its own proteins (electron carriers and enzymes) (10%) + can also self-replicate as chloroplast has its own DNA polymerase

   • RNA polymerase and 70S ribosomes which allow chloroplasts to synthesise their own proteins

   • enzymes required for Calvin cycle/dark reaction of photosynthesis (e.g., Rubisco) to synthesise organic compounds (e.g., carbohydrates)

   • starch grains to store excess glucose

function of chloroplast

1. carry out photosynthesis

   • absorb light energy to synthesise ATP and NADPH (light reaction) which are used to synthesise organic compounds (e.g. sugars such as glucose)

2. store food reserves (e.g. as starch grains)

chloroplast envelope

structure: bounded by double membrane → both outer and inner membrane form smooth continuous boundaries, and the inner membrane is impermeable to ions

function: separates organelle from the cell and allows for
compartmentalisation

thylakoids (third membrane system) → structure and function

1. thylakoid membrane forms a network of flattened discs known as
   thylakoids, and are usually stacked to form grana (singular: granum), and can be joined by intergranal lamella (plural: lamellae)

   • a site where light reactions occur

   • extensive thylakoid membrane provides a large surface area for attachment of:

     • photosystems containing photosynthetic pigments (e.g. chlorophyll, carotenoids) for absorption of light energy during light reaction of photosynthesis

     • electron carriers

     • enzymes (NADP+ reductase)

     • ATP synthase (also known as stalked particles)

   • photosystems and electron carriers are closely located and arranged in a sequential order for electrons to be passed down the chain of electron carriers of decreasing energy levels

     • releases energy to pump protons (H+) into the thylakoid space (active transport) → increases efficiency of photophosphorylation

   • the phospholipid bilayer is impermeable to H+, thus
     allowing accumulation of H+ in the thylakoid space to create a steep proton gradient

2. inside the thylakoid is the thylakoid space

   • important for creating a steep proton gradient for
     chemiosmosis

stroma → structure and function

1. contains many copies of circular chloroplast DNA

   • genes code for its own proteins such as electron carriers and enzymes needed for dark reactions

   • as chloroplast has its own DNA polymerase, the circular DNA can self-replicate

2. contains 70S ribosomes

   • allow chloroplast to carry out protein synthesis independently of the cell

3. gel-like medium containing soluble enzymes, organic acids,
   lipids, sugars + contain varying concentrations of ATP/ADP, NADPH/NADP

   • site where dark reaction / Calvin Cycle occurs

     • requires enzymes (e.g. Rubisco) to synthesise organic compounds (e.g. carbohydrates)

   • starch grains to store excess glucose as starch

absorption spectrum

absorption spectrum: graph of amount of light energy absorbed for each pigment at each wavelength (λ)

• various pigments (e.g. chlorophyll a, chlorophyll b, β-carotenes) absorb light energy from a range of 400 to 700nm

  • blue and red light brings about high rate of photosynthesis

• single chloroplast contains thousands of photosystems → each is a cluster of photosynthetic pigments embedded in thylakoid membranes of chloroplasts

• all wavelengths of visible white light, except green, are absorbed

  • chlorophylls a and b absorb light energy from both blue (400-500nm) and red (600-700nm) regions

  • β-carotene and xanthophyll absorb light energy from blue (400-500nm)

action spectrum

action spectrum: graph of rate of photosynthesis occurring at each wavelength (λ)

• there is a close similarity between absorption and action spectra ⇒ photosynthetic pigments are responsible for absorption of light energy

• the relationship between both spectra is not directly proportional

roles of pigment

• photosynthetic pigments are found in photosystems embedded in thylakoid membrane

• two types of photosystems (PS):

  • PS I: P700 reaction centre (special chlorophyll a absorbs light energy best at 700nm)

  • PS II: P680 reaction centre (special chlorophyll a absorbs light energy best at 680nm)

structure of photosystems → each consists of:

1. a light harvesting complex (LHC)

   • consists of accessory pigments (e.g. chlorophyll a, chlorophyll b, carotenoids)

   • which absorb light energy from 400nm to 700nm and transfer energy to the reaction centre

2. a reaction centre (RC) surrounded by LHC

   • consists of primary pigments (special chlorophyll a) whose electrons are displaced during photoactivation (activated by light)

   • when sufficient energy is absorbed, an electron of
     special chlorophyll a is boosted to very high energy
     level and is displaced

   • the displaced electron is accepted by electron acceptor and pass down electron carriers of decreasing energy levels in the Electron Transport Chain (ETC)

overview of photosynthesis

• photosynthesis is a two-stage process

  1. light reaction (light dependent reaction) / photophosphorylation

     • dependent on light intensity

  2. dark reaction (light independent reaction) / calvin cycle

     • independent on light intensity

     • dependent on temperature, substrate concentration, enzyme concentration, pH (enzyme-controlled)

6CO₂ + 6H₂O → C₆H₁₂O₆ + 6O₂​

light reaction → photophosphorylation

• occurs at thylakoid membrane (including grana and intergranal lamellae)

• light energy is required (hence light dependent reaction) for photoactivation of special chlorophyll a and photolysis of water

  • light energy is converted to chemical energy stored in products NADPH and ATP, which are required for dark reaction

• O₂ is a by-product released during photolysis of water

requirements for light reaction

• for non-cyclic photophosphorylation

  • light energy

  • NADP⁺

  • H₂O

• for cyclic photophosphorylation

  • light energy

products for light reaction

• for non-cyclic photophosphorylation

  • ATP for dark reaction (Calvin Cycle)

  • NADPH for dark reaction (Calvin Cycle)

  • O₂ (by-product)

• for cyclic photophosphorylation

  • ATP for dark reaction (Calvin Cycle)

non-cyclic photophosphorylation

• occurs all the time when light energy is present ⇒ makes products used in Calvin Cycle

• synthesise of ATP and NADPH via photophosphorylation

1. at photosystem II (PSII), light energy is absorbed by photosynthetic (accessory) pigments of light harvesting complex (LHC)

2. energy is passed to neighbouring pigments until it reaches the pair of special chlorophyll a in the reaction centre (RC)

3. photoactivation of special chlorophyll a at reaction centre results in electron displaced from the pair of special chlorophyll a (oxidised)

4. photolysis of water occurs (only in PSII) (in thylakoid space) to replenish displaced electrons from PSII

   • enzyme for photolysis is located at PSII

   • H+ / protons accumulates in the thylakoid space ⇒ steep proton gradient

   • oxygen is released as a by-product [2H₂O → 4H⁺ + 4e^- + O₂] ⇒ diffuse out

5. electrons (from special chlorophyll a at reaction centre) are passed down electron carriers of decreasing energy levels in the electron transport chain (ETC) from PS II to PS I → unidirectional path

   • [further explanation] electron carriers (involved in a series of redox reactions) are found in thylakoid membrane arranged in order of decreasing energy levels

   • electron displaced from PS II is first accepted by primary electron acceptor, and the electron is then passed down electron carriers of the first Electron Transport Chain (ETC) between PS II and PS I, until it reaches the pair of special chlorophyll a in the Reaction Centre of PS I

6. energy released from flow of electrons is used to pump H+ from stroma (across thylakoid membrane) into thylakoid space, and this creates a steep proton gradient

   • [further explanation] concentration of H+ in thylakoid space (pH 4) is greater than that in stroma (pH 8)

   • thylakoid membrane is impermeable to H+ due to hydrophobic core of phospholipid bilayer

7. photoactivation occurs simultaneously in PS I (i.e., same processes 1 to 3; 5 and 6)

8. electron displaced from PS I is passed down electron carriers of second ETC

9. electron is accepted by final electron acceptor NADP⁺ to form NADPH (reduced NADP), catalysed by enzyme NADP⁺ reductase [NADP⁺ + 2H⁺ + 2e^- → NADPH + 2H⁺]

   • diffusion of H+ from thylakoid space into stroma through hydrophilic channel of ATP synthase (stalked particle) down concentration gradient releases energy, which is coupled to ATP synthesis, catalysed by ATP synthase via chemiosmosis

   • [further explanation of chemiosmosis] chemiosmosis refers to the diffusion of H+ from thylakoid space into stroma
     through hydrophilic channel of ATP synthase (stalk particle) down concentration gradient releases energy which is coupled to ATP synthesis, catalysed by ATP synthase

   • the proton gradient is created using energy released when electrons are passed down electron carriers of decreasing energy level and photolysis of water in the thylakoid space

cyclic photophosphorylation → only at PS1

• occurs when more ATP is needed ⇒ only ATP is synthesised (no NADPH is produced)

• PS I is both the electron donor and acceptor

  • PS II is not involved

  • no photolysis of water

1. at PSI, light energy is absorbed by photosynthetic (accessory) pigments of light harvesting complex (LHC)

2. energy is passed to neighbouring pigments until it reaches the pair of special chlorophyll a in reaction centre (RC)

3. photoactivation of special chlorophyll a at RC results in electron displaced from the pair of special chlorophyll a

4. electron is passed down electron carriers of decreasing energy levels from PS I to the ETC between PS II and PS I, and back to PS I, hence, NADPH is not formed [no water is needed ⇒ no photolysis]

5. energy released from flow of electrons is used to pump H+ from stroma (across thylakoid membrane) into thylakoid space, and this creates a steep proton gradient

6. diffusion of H+ from thylakoid space into stroma through hydrophilic channel of ATP synthase (stalked particle) down concentration gradient releases energy, which is coupled to ATP synthesis, catalysed by ATP synthase via chemiosmosis

non-cyclic photophosphorylation VS cyclic photophosphorylation

calvin cycle

• occurs in stroma

• light energy is not directly involved in the reaction ⇒ light independent

• occurs all the time (even in absence of light) if there are products of light reaction (ATP and NADPH) available to fix CO₂ and synthesise carbohydrates & other organic molecules [incorporates CO₂ into organic molecules, converted to sugar]

requirements for calvin cycle

• from light reaction: 2 high energy chemical molecules which drive
  Calvin cycle

  • ATP for carbon reduction and RuBP regeneration

  • NADPH for carbon reduction

• from air:

  • CO₂ for carbon fixation

• within stroma of chloroplast:

  • Ribulose-1,5-bisphosphate (RuBP)

compounds exiting the calvin cycle

• ADP

• NADP⁺

• Glyceraldehyde-3-phosphate 3 also known as triose phosphate (TP)/GALP/G3P

processes in calvin cycle

carbon (CO₂) fixation

1. Rubisco (Ribulose-1,5-bisphosphate carboxylase-oxygenase) catalyses CO₂ fixation

2. CO₂ combines with RuBP (5C) to form an unstable 6C compound, which breaks down into 2 molecules of phosphoglycerate4 (PGA / GP) (3C)

PGA reduction

3. Phosphoglyceric acid (PGA) (also known as glycerate-3-phosphate) is phosphorylated and then reduced to glyceraldehyde-3-phosphate (or triose phosphate [TP] or GALP or G3P or PGAL) using ATP (provides energy) and NADPH (oxidised) from light reaction

regeneration of RuBP

4. Glyceraldehyde-3-phosphate (TP) is phosphorylated using ATP from light reaction and rearranged to regenerate ribulose-1,5-bisphosphate (RuBP) for Calvin Cycle to continue to occur

   • 5 TP (3C) molecules rearrange to form 3 RuBP (5C), which require total of 3 ATP

5. one glyceraldehyde-3-phosphate (TP) exits the cycle to form organic compounds (e.g. carbohydrates, amino acids, fatty acids)

   • [further explanation] 2 TP (3C) molecules combine to form glucose (6C), which is used to form starch for storage

important pointers for photosynthesis experiments

1. rate of photosynthesis can be measured by:

   • volume of carbon dioxide taken in per unit time → rate of substrate being used up

   • volume of oxygen evolved per unit time → rate of product being formed

   • increase in dry mass per unit time → rate of product being formed – carbon fixation

2. when the effect of different experimental conditions (e.g., light intensity) on the rate of photosynthesis is investigated, it is best to use the same individual plant

3. when the rates of photosynthesis of different plants are compared, calculate the rate of photosynthesis per unit time and per unit mass of plant (or per unit surface area of leaf) before
   comparison

4. remember to take into consideration the rate of respiration before calculation of the rate of photosynthesis

   • products of photosynthesis (carbohydrates, oxygen) are used in respiration

   • product of respiration (carbon dioxide) is used for photosynthesis

limiting factor

limiting factor: a factor that is nearest to its minimum value and directly increases the rate of reaction when its quantity is changed

• typical graph of the effect of a factor affecting rate of photosynthesis

  • all factors (e.g., light intensity) affecting the rate of photosynthesis will have their effects presented
    in the form of a graph

light intensity

• light energy is required for photoactivation of special chlorophyll a and photolysis of water in light reaction/photophosphorylation in order to synthesise NADPH and ATP for Calvin Cycle

• light intensity is usually not a major limiting factor for most plants (except for shade plants) unless it is during sunrise and sunset

  • light intensity on a clear summer day → about 100 000 lux

  • light saturation for C3 plants → about 10 000 lux (10% of full sunlight

• very high light intensity may damage chlorophyll (photobleaching) which reduces the rate of photosynthesis

  • plants exposed to such conditions (e.g., desert plants) usually have thick cuticles and hairy leaves

to describe light intensity graph

• A: rate of photosynthesis increases steeply (and linearly)

  • light intensity is the limiting factor

  • as light intensity increases, more light energy is absorbed by photosynthetic pigments

  • rate of photoactivation of special chlorophyll a increases, and the rate of light reaction and photosynthesis increases

• B: rate of photosynthesis increases gradually → slows down

  • another factor is becoming the limiting factor

• C: rate of photosynthesis remains constant / is at its maximum

  • light saturation point is reached

  • light intensity is no longer the limiting factor; another factor is the limiting factor [it could be CO2 or
    temperature]

compensation point → light intensity

compensation point: light intensity whereby rate of photosynthesis equals rate of respiration

• there is no net gaseous exchange between the plant and its environment

  • all CO₂ released during respiration are used in photosynthesis

  • O₂ produced during photosynthesis are used for respiration

• no net increase in dry mass and no growth of plants

• compensation point is reached at low light intensity, usually during sunrise and sunset

carbon dioxide concentration

• CO₂ is required for carbon fixation during dark reaction

• CO₂ concentration is a major limiting factor in photosynthesis since its concentration in the atmosphere is about 0.04%

  • as carbon dioxide concentration increases, the rate of synthesis of carbohydrates via dark reaction / Calvin Cycle increases until limited by other factors

  • [FYI] however, increased atmospheric CO₂ may result in fewer stomata formed

temperature

• temperature affects the rate of enzymatic reaction

  • especially for enzymes involved in Calvin Cycle (dark reaction)

  • enzymes (e.g., NADP+ reductase, ATP synthase) in light reaction are also affected

• for every 10°C increase in temperature, up till its optimum temperature, the rate of photosynthesis doubles

• beyond the optimum temperature, the enzymes denature

other factors affecting rate of photosynthesis

1. effect of light quality (wavelength)

   • rate of photosynthesis is higher when blue or red wavelengths are absorbed

2. effect of oxygen concentration

   • Rubisco has a binding affinity for both CO₂ and O₂→ both can bind to Rubisco

   • at high CO₂ concentration, CO₂ binds at the active site of rubisco for carbon fixation

   • at low CO₂ concentration, O₂ successfully competes with CO₂ for the active site of Rubisco ⇒ rate of carbon fixation is reduced

3. effect of availability of water

   • only less than 1% of water absorbed by roots are used in photosynthesis, hence availability of water in the soil is unlikely to affect the rate of photosynthesis

   • however, when there is very high rate of transpiration (greater than uptake of water), it results in the closure of stomata, in order to reduce water loss

   • the rate of gaseous exchange decreases, resulting in decrease in the rate of carbon fixation

4. effect of chlorophyll concentration

   • chlorophyll concentration is usually not a limiting factor

   • decrease in chlorophyll concentrations causes leaves to turn yellow ⇒ rate of photosynthesis decreases

   • reasons:

     • magnesium and nitrogen deficiency → both are part of structure of chlorophyll

     • lack of light → light is required for final stage of chlorophyll synthesis

     • disease or ageing

orientation of ATP synthase in mitochondrion and chloroplast:

similarities between photophosphorylation VS oxidative phosphorylation

1. electrons passed down electron carriers of decreasing energy level in electron transport chain

2. pumping of H+ across membrane to create steep proton gradient

3. diffusion of H+ via hydrophilic channel of ATP synthase (stalked particle) to synthesise ATP

differences between photophosphorylation VS oxidative phosphorylation

feature of comparison	photophosphorylation	oxidative photophosphorylation
location	takes place in the thylakoid of chloroplasts	takes place in the inner mitochondrial membrane
source of electrons	[non-cyclic] water [cyclic] PS 1	NADH FADH2
final electron acceptor	[non-cyclic] NADP+ [cyclic] PS 1	O2
products formed	3 products are formed: ATP, NADPH, O2	2 products are formed: ATP, H2O
requirement of light energy	required for photolysis of water	not required
source of energy	light energy	oxidation of glucose provides source of energy
direction of H+ pumped to generate steep proton gradient	H+ pumped from stroma to thylakoid space	H+ pumped from matrix to intermembrane space
direction of H+ diffusion to synthesise ATP	diffusion from thylakoid space to stroma	diffusion from intermembrane space to matrix

similarities between Calvin Cycle VS Krebs Cycle

involve cyclic pathways / regeneration of intermediates

differences between Calvin Cycle VS Krebs Cycle

features of comparison	Calvin Cycle	Krebs Cycle
location	stroma of chloroplast	matrix of mitochondrion
(starting) intermediate regenerated	RuBP is regenerated	oxaloacetate is regenerated
compounds exiting cycle	TP/GALP/G3P, NADP+, ADP	NADH, FADH2, ATP
involvement of CO2	CO2 is fixed by rubisco	CO2 is released during oxidative decarboxylation
role of electron/hydrogen carrier	NADPH provides reducing power for carbon reduction	NADH is a product formed during oxidation of acetyl-CoA
role of ATP	phosphorylation of glycerate-3-phosphate (PGA) to form glyceraldehyde-3 phosphate (TP) regeneration of RuBP	synthesise ATP by substrate level phosphorylation
type of metabolic process/enzyme reaction	anabolic reaction → carbon is fixed to synthesise organic compounds	catabolic reaction→ carbon is removed as CO2 from organic compound

similarities between chloroplast VS mitochondrion

1. both chloroplasts and mitochondria have an outer and inner membrane

2. both organelles, outer membranes form a smooth and continuous boundary

3. both organelles have circular DNA

4. both organelles have 70S ribosomes

5. both have electron carriers embedded in the internal membrane system → inner membrane of mitochondria and thylakoid membrane of chloroplast

6. both have ATP synthase embedded in the internal membrane system → inner membrane of mitochondria and thylakoid membrane of chloroplast

7. both have an extensive system of membrane inside

differences between chloroplast VS mitochondrion

feature of comparison	chloroplast	mitochondrion
folding of inner membrane to form cristae	no	yes
presence of thylakoid/grana	yes	no
presence of photosystems/ chlorophyll embedded in thylakoid membrane	yes	no
presence of starch grains	yes	no
direction of projection of ATP synthase → stalked particle	project outwards into the stroma	project inwards into matrix

similarities between photosynthesis VS respiration

1. require organelles

   • chloroplasts for photosynthesis

   • mitochondria for respiration

2. energy-converting process

3. cyclic pathway involved

   • Calvin Cycle for photosynthesis

   • Krebs Cycle for respiration

4. require Electron Transport Chain for chemiosmosis to occur, and the ETC is found on:

   • thylakoid membrane for photosynthesis

   • inner mitochondrial membrane for respiration

5. ATP synthesis via ATP synthase/phosphorylation occurs

differences between photosynthesis VS respiration

feature of comparison	photosynthesis	respiration
organelle where process occurs	chloroplast	mitochondrion
type of cells	cells containing photosynthetic pigments (chlorophyll)	all cells (all the time)
involvement of light	yes for light reaction	no
use of energy	energy is accumulated and used to synthesise carbohydrates and stored in carbohydrates	energy is used to synthesise ATP
dry mass of cell	increase	decrease
involvement of oxygen	released as by-product of light reaction	used as final electron acceptor in oxidative phosphorylation
involvement of carbon dioxide	fixed during carbon fixation in Calvin Cycle to synthesise carbohydrates	released during oxidative decarboxylation in link reaction and Krebs Cycle
involvement of water	used for photolysis of water in light reaction	released after final electron acceptor oxygen accepts electrons in oxidative phosphorylation
type of metabolic process/enzyme reaction	anabolic → carbon is fixed to synthesise organic compounds	catabolic → carbon is removed as CO2CO_2CO2​ from organic compound)