Chloroplasts and Photosynthesis

• Chloroplasts are the organelles in plant cells where photosynthesis occurs.
• Chloroplasts are surrounded by a double-membrane envelope, and each membrane is a phospholipid bilayer.
• The stroma is the fluid-filled space within chloroplasts where the light-independent stage of photosynthesis occurs.
• Thylakoids are flattened fluid-filled sacs in the stroma where the light-dependent stage of photosynthesis takes place.
• Grana are stacks of thylakoids, connected by stroma lamellae.
• Grana membranes create a large surface area, increasing the number of light-dependent reactions.
• The stroma includes 70S ribosomes, DNA loops, and starch grains; the DNA codes for some chloroplast proteins that are produced at the 70S ribosomes.
• Sugars formed during photosynthesis are stored as starch inside starch grains.

Stages of Photosynthesis

• Light-dependent stage occurs in the thylakoids.
• Light-independent occurs in the stroma.
• During the light-dependent stage, reduced NADP is produced when hydrogen ions combine with NADP using electrons from photolysis.
• ATP is produced from ADP and P through photophosphorylation, utilizing the proton gradient from photolysis.
• Energy from ATP and hydrogen from reduced NADP are then passed to the light-independent stage.
• During the light-independent reactions (Calvin cycle), energy and hydrogen are used to produce organic molecules like starch, sucrose, and cellulose.

Thylakoids & Stroma

• Plant cells contain chloroplasts, which are the site of photosynthesis.
• Chloroplasts are filled with fluid known as the stroma.
• The membrane system in the stroma are thylakoids, which stack to form grana.
• Light-dependent photosynthesis occurs in the thylakoid membranes and thylakoid spaces.
• Thylakoid membranes contain pigments, enzymes, and electron carriers for light-dependent reactions.
• Grana membranes create a large surface area, allowing for more light-dependent reactions.
• The membrane system provides pigment molecules in an arrangement that maximizes light absorption.
• Pigment molecules are arranged in light-harvesting clusters known as photosystems.
• In a photosystem, pigment molecules in the thylakoid membrane each pass energy down to the next molecule until it reaches the primary pigment reaction centre.
• The stroma is the fluid that fills the chloroplasts and surrounds thylakoids and contains CO2, sugars, enzymes, and other molecules.
• The stroma is the site of the light-independent stage of photosynthesis.

Chloroplast Pigments

• Photosynthetic pigments within the thylakoids absorb different light wavelengths.
• Thylakoids stack to form grana.
• The thylakoid membrane system provides a large arrangement of pigment molecules, ensuring light is absorbed.
• Pigment molecules are arranged in photosystems
• In a photosystem, pigment molecules are arranged in funnel-like structures in the thylakoid membrane, where energy is passed down the line until it reaches the primary pigment reaction center.
• Light-dependent photosynthesis occurs in the thylakoid membranes and thylakoid spaces.
• Thylakoid membranes contain pigments, enzymes, and electron carriers required for the light-dependent reactions.
• There are two groups of pigments; chlorophylls (chlorophyll a and b) and carotenoids (carotene and xanthophyll).
• Chlorophylls absorb wavelengths in the blue-violet and red regions, reflecting green light.
• Carotenoids absorb wavelengths of light mainly in the blue-violet region.

Absorption Spectra & Action Spectra

• An absorption spectrum shows the absorbance of different light wavelengths by a pigment.
• Chlorophylls absorb wavelengths in the blue-violet and red.
• Carotenoids absorb wavelengths of light mainly in the blue-violet.
• An action spectrum shows the rate of photosynthesis at different wavelengths of light.
• The rate of photosynthesis is highest in the blue-violet and red, which are the wavelengths that plants can absorb.
• There is a strong correlation between the cumulative absorption spectra of pigments and the action spectrum.
• Both graphs have two main peaks at the blue-violet and red regions and a trough in the green-yellow region.

Chromatography of Chloroplast Pigments

• Chromatography is a technique used to separate mixtures.
• The mixture is dissolved in a fluid/solvent (mobile phase) and passed through a static material (stationary phase).
• Different components travel through at different speeds, causing separation.
• A retardation factor (Rf) can be calculated: distance travelled by component ÷ distance travelled by solvent.
• Two common techniques for separating photosynthetic pigments are paper chromatography (cellulose) and thin-layer chromatography (silica gel).
• Chromatography can identify chloroplast pigments from a leaf, each with a unique Rf value that indicates how far the dissolved pigment travels.
• A smaller Rf value indicates the pigment is less soluble and larger.
• Carotenoids have the highest Rf values.
• Chlorophyll B has a much lower Rf value.
• Chlorophyll A has an Rf value between the two.

Photophosphorylation

• The thylakoid membrane is the site of the light-dependent stage of photosynthesis.
• During the light-dependent stage, light energy breaks down water, producing hydrogen ions, electrons, and oxygen.
• A proton gradient forms as a result of the photolysis of water.
• Electrons travel through an electron transport chain of proteins within the membrane.
• Reduced NADP (NADPH) is produced when hydrogen ions in the stroma and electrons from the electron transport chain combine with the carrier molecule NADP.
• ATP is produced during a process known as photophosphorylation using the proton gradient and ATP synthase.
• Photophosphorylation can be cyclic or non-cyclic, based on electron flow in photosystem I (PSI) or photosystem II (PSII).
• Cyclic photophosphorylation (PSI only) and non-cyclic photophosphorylation (both PSI and PSII) occur.
• Photosystems are pigment collections that absorb light energy and transfer the energy onto electrons, each one contains a primary pigment.
• Photosystem II has a primary pigment called P680 that absorbs light at 680nm, and is where the photolysis of water takes place, at the beginning of the electron transport chain.
• Photosystem I has a primary pigment called P700 that absorbs light at 700nm, in the middle of the chain.
• The energy carried by the ATP is then used during the light-independent reactions of photosynthesis.
• Cyclic photophosphorylation involves photosystem I (PSI) only.
• Light is absorbed by photosystem I and passed to the primary pigment (P700).
• An electron in the chlorophyll molecule is excited to a higher energy level, and is emitted from the chlorophyll molecule in a known process, photoactivation.
• The excited electron is captured by an electron acceptor, transported via an electron transport chain.
• As electrons pass through the electron transport chain, they provide energy to transport protons from the stroma to the thylakoid lumen via a proton pump.
• The proton build-up in the thylakoid lumen drives the synthesis of ATP by chemiosmosis.
• Chemiosmosis is the movement of (protons) down their concentration gradient, and the released energy is used by ATP synthase to synthesise ATP.
• The ATP passes to the light-independent reactions.
• Non-cyclic photophosphorylation involves both photosystem I (PSI) and photosystem II (PSII).
• Light is absorbed by photosystem II and passed to the primary pigment (P680).
• An electron in the chlorophyll molecule is excited to a higher energy level, and is emitted from the chlorophyll molecule in the known process, photoactivation.
• The excited electron is passed down a chain of electron carriers where ATP is synthesized from ADP using chemiosmosis.
• The ATP then goes to the light-independent reactions.
• Photosystem II contains a water-splitting enzyme that catalyses photolysis: H2O → 2H+ + 2e- + 1/2O2.
• As the excited electrons leave the primary pigment of photosystem II, they are replaced by electrons from photolysis.
• At the same time, photoactivation of electrons also occurs in photosystem I.
• The excited electrons from photosystem I pass along an electron transport chain.
• These electrons combine with hydrogen ions and NADP to give reduced NADP: 2H+ + 2e + NADP → reduced NADP.
• The reduced NADP (NADPH) then passes to the light-independent reactions for carbohydrate synthesis.
• During photophosphorylation, energetic electrons are captured by an electron acceptor .
• These are passed along a chain of electron carriers.
• The electron carriers are alternately reduced and oxidised.
• The excited electrons release energy as they pass through the chain.
• The released energy actively transports protons across the thylakoid membrane, from the stroma to the thylakoid lumen through a “proton pump".
• This creates a proton gradient, with a high proton concentration in the thylakoid lumen and a low concentration in the stroma.
• Protons return to the stroma by facilitated diffusion through transmembrane ATP synthase in a process known as chemiosmosis.
• This process provides the energy needed to synthesise ATP by adding an inorganic phosphate group (Pi) to ADP (ADP + P; → ATP).
• This whole process is known as photophosphorylation as light provides the initial energy source for ATP synthesis.
• After being passed down the electron transport chain, the de-energised electrons from photosystem II are taken up by photosystem I

The Calvin Cycle

• Energy from ATP and hydrogen from reduced NADP are passed from the light-dependent stage to the light-independent stage of photosynthesis
• The energy and hydrogen are used during the light-independent reactions (known collectively as the Calvin cycle) to produce complex organic molecules like: Starch (for storage), Sucrose (for translocation around the plant), and Cellulose (for making cell walls)
• This stage of photosynthesis does not, in itself, require energy from light and can therefore take place in light or darkness.
• The Calvin cycle cannot continue indefinitely in darkness, as it requires inputs of ATP and reduced NADP from the light-dependent stage.
• There are three main steps within the Calvin cycle:
• Rubisco catalyses the fixation of carbon dioxide by combination with a molecule of ribulose bisphosphate (RuBP), to yield two molecules of glycerate 3-phosphate (GP).
• GP is reduced to triose phosphate (TP) in a reaction involving reduced NADP and ATP
• RuBP is regenerated from TP in reactions that use ATP
• Carbon dioxide combines with a five-carbon (5C) sugar known as ribulose bisphosphate (RuBP).
• An enzyme called rubisco catalyses this reaction.
• The resulting six-carbon (6C) compound is unstable and splits in two.
• This gives two molecules of a three-carbon (3C) compound known as glycerate 3-phosphate (GP).
• The carbon dioxide has been 'fixed' (it has been removed from the external environment and has become part of the plant cell).
• Glycerate 3-phosphate (GP) is not a carbohydrate but the next step in the Calvin cycle converts it into one.
• Energy from ATP and hydrogen from reduced NADP, produced during the light-dependent stage of photosynthesis are used to reduce glycerate 3-phosphate (GP) to a phosphorylated three-carbon (3C) sugar known as triose phosphate (TP).
• One-sixth of the triose phosphate (TP) molecules are used to produce organic molecules needed by the plant; Triose phosphates can condense to become hexose phosphates (6C), which can be used to produce starch, sucrose or cellulose.
• Triose phosphates can be converted to glycerol and glycerate 3-phosphates to fatty acids, which join to form lipids for cell membranes.
• Triose phosphates can be used in the production of amino acids for protein synthesis
• Five-sixths of the triose phosphate (TP) molecules are used to regenerate ribulose bisphosphate (RuBP).
• This process requires ATP.
• Intermediate molecules of the Calvin cycle (such as glycerate 3-phosphate and triose phosphate) are used to produce other molecules.
• Glycerate 3-phosphate (GP) is used to produce some amino acids.
• Triose phosphate (TP) is used to produce:
• Hexose phosphates (6C) to produce starch, sucrose or cellulose
• Lipids for cell membranes
• Amino acids for protein synthesis

Limiting Factors of Photosynthesis

• Plants need several factors for photosynthesis to occur: photosynthetic pigments, a supply of carbon dioxide, a supply of water, light energy, a suitable temperature
• If there is a shortage of any of these factors, photosynthesis cannot occur at its maximum possible rate
• The main external factors that affect the rate of photosynthesis are: light intensity, carbon dioxide concentration, and temperature
• These are known as limiting factors of photosynthesis
• If any one of these factors is below the optimum level for the plant, its rate of photosynthesis will be reduced, even if the other two factors are at the optimum level
• Changes in light intensity, carbon dioxide concentration and temperature are all limiting factors that affect the rate of photosynthesis
• The rate of photosynthesis increases as light intensity increases: The greater the light intensity, the more energy supplied to the plant and therefore the faster the light-dependent stage of photosynthesis can occur, and producing more ATP and reduced NADP for the Calvin cycle.
• If light intensity continues to increase, the rate of photosynthesis reaches a plateau where light intensity is not longer a limiting factor and another factor is limiting.
• The factors that could be limiting the rate when the line on the graph is horizontal include temperature or not enough carbon dioxide.
• The rate of photosynthesis increases as carbon dioxide concentration increases: It is required for the light-independent stage of photosynthesis with the five-carbon compound ribulose bisphosphate (RuBP).
• However, it increases to a certain point and then continues.
• As temperature increases, the rate of photosynthesis increases to a point until the enzymes begin to denature and the rate of reaction decreases.
• For photosynthesis, temperature has no significant effect on the light-dependent reactions, as these are driven by energy from light rather than the kinetic energy of the reacting molecules
• However, the Calvin cycle is affected by temperature, as the light-independent reactions are enzyme-controlled reactions (eg. rubisco catalyses the reaction between CO2 and the five-carbon compound ribulose bisphosphate)

Investigating the Rate of Photosynthesis: Redox Indicators

• The light-dependent reactions of photosynthesis take place in the thylakoid membrane and involve the release of high-energy electrons from chlorophyll a molecules.
• These electrons are picked up by electron acceptors and then passed down the electron transport chain.
• However, if a redox indicator (such as DCPIP or methylene blue) is present, the indicator takes up the electrons instead.
• This causes the indicator to change color:
  • DCPIP: oxidized (blue) becomes reduced (colorless) after accepting e-
  • Methylene blue: oxidized (blue) becomes reduced (colorless) after accepting e-
• The color of the reduced solution may appear green because the chlorophyll has a green color.
• The rate at which the redox indicator changes color from its oxidized state to its reduced state can be used as a measure of the rate of photosynthesis.
• When light is at a higher intensity, or at more preferable light wavelengths, the rate of photoactivation of electrons is faster, therefore the rate of reduction of the indicator is faster

Investigating the Rate of Photosynthesis: Aquatic Plants

• Investigations to determine the effects of light intensity, carbon dioxide concentration and temperature on the rate of photosynthesis can be carried out using aquatic plants:
  • Light intensity: change the distance (d) of a light source from the plant (light intensity is proportional to 1/d2)
  • Carbon dioxide concentration: add different quantities of sodium hydrogencarbonate (NaHCO3) to the water surrounding the plant, this dissolves to produce CO2
  • Temperature: place the boiling tube containing the submerged plant in water baths of different temperature
• Ensure the water is well aerated before use by bubbling air through it, to ensure oxygen gas forms bubbles.
• Ensure the plant has been well illuminated before use
• Set up the apparatus in a darkened room with sodium hydrogencarbonate solution
• Cut the stem of the pondweed cleanly just before placing into the boiling tube
• Measure the volume of gas collected in the gas-syringe in a set period of time
• Change the independent variable and repeat the method
• Record the results in a table and plot a graph of volume of oxygen