After studying this chapter, the reader should be able to:

• Describe the compartments of the chloroplast, especially the thylakoid membrane and thylakoid space.
• Discuss the structure and function of the photosystems.
• Discuss the carbon fixation reactions of photosynthesis.
• Give examples of genes expressed from the chloroplast genome.

1. General information
1. In plants, chloroplasts are the site of photosynthesis
2. The chloroplast organelle is composed of three membrane layers: the outer and inner chloroplast membranes and the thylakoid membrane; this structure creates three compartments: the intermembrane space, the stroma and the thylakoid space
3. Light harvesting reactions, electron transport, and ATP synthesis take place on the thylakoid membrane
4. Chloroplast DNA and protein synthesis machinery are located in the stroma
2. Discovery
1. Because of their large size and green color, chloroplasts in plant cells were identified by early microscopists, including Anthony van Leeuwenhoek, in the 17th century
2. In 1682, Nehemiah Grew described green precipitates in leaves; this event is considered the first report of the existence of chloroplasts
3. The light-dependent production of oxygen by plants was discovered by Joseph Priestley in 1771
1. A mouse in an enclosed glass jar in which there was a burning candle died, but when a plant was also enclosed within the jar, it lived
2. Priestley wrote: "I have been so happy as by accident to hit upon a method of restoring air which has been injured by the burning of candles and to have discovered at least one of the restoratives which Nature employs for this purpose. It is vegetation."
3. Burning depletes oxygen in air in the jar, and the plant generates oxygen by photosynthesis
3. Structure and composition
1. Chloroplasts are members of the plastid family of subcellular organelles and are specific to eukaryotes
1. Chloroplasts are 4-6 m m in diameter and 5-10 m m in length
2. Chloroplasts are present only in special photosynthetic structures, namely leaves
3. A typical leaf cell may have 50-100 chloroplasts
2. In algae and photosynthetic protozoans, chloroplasts are more variable in number and in shape
1. Spirogyra has 1 or 2 ribbon-shaped, helical chloroplasts
2. Chlamydomonas has 1 large cup-shaped chloroplast
3. The plastid family has many members
1. Chloroplasts (green) contain pigments called chlorophylls
2. Chromoplasts (yellow or orange) contain pigments called carotenoids
3. Leukoplasts, found in embryonic and germ cells, are colorless
4. Amyloplasts contain starch granules for storage
5. Etioplasts are immature chloroplast of plants grown in the dark
6. All of these plastids are derived from proplastids (immature plastids)
4. A chloroplast is divided into three compartments (the intermembrane space, the stroma and the thylakoid space) by three membranes (the outer chloroplast membrane, the inner chloroplast membrane and the thylakoid membrane)
5. The outer and inner chloroplast membranes form the outer envelope of the organelle, whereas the thylakoid membrane forms a complex lamellar system with stacked membrane discs known as grana (singular, granum)
1. The outer envelope membranes contain little protein
1. The ratio is 70% lipid to 30% protein
2. As in mitochondria, the outer chloroplast membrane is highly permeable and the inner membrane is selectively permeable, with special carrier proteins called translocases
3. Glycolipid synthesis enzymes are located in the outer envelope membranes
2. The thylakoid membrane is the site of photosynthesis
1. The folding of the thylakoid membrane to form grana greatly increases the surface area
2. It is estimated that the surface area of the photosynthetic membranes of a typical leaf cell is 600X greater than the cell's surface
3. As a site for the electrochemical proton gradient, the thylakoid membrane is ion-impermeable, resembling the inner mitochondrial membrane
4. Special glycolipids, monogalactosyldiacylglycerol and digalactosyldiacylglycerol (constituting to 70% of thylakoid lipid), may be involved for thylakoid membrane function
5. The thylakoid membrane is the site for photosystems I and II, the electron transport carriers, and the chloroplast ATP synthase (also called CF₀CF₁ ATPase)
6. Thus, like the inner mitochondrial membrane, protein is abundant in the thylakoid membrane, with a lipid:protein ratio of 30:70
6. The stroma of the chloroplasts is analogous to the matrix of the mitochondria
1. As in mitochondria, chloroplasts contain their own DNA and ribosomes; these are located in the stroma
2. Calvin cycle enzymes, fatty acid synthase and enzymes for amino acid synthesis are also located in the chloroplast stroma

1. General information
1. Photosynthesis is the process by which plants and some bacteria use the energy of sunlight to drive the synthesis of organic molecules from water and carbon dioxide
2. Photosynthesis takes place in the plant chloroplasts; the process can be separated into light-harvesting light reactions and light-independent dark reactions of carbon fixation
3. Photosystems I and II, electron transport carriers, and ATP synthase are all needed for photosynthesis
4. Both cyclic and noncyclic photophosphorylations are possible
2. The light reactions
1. The photosynthetic electron transfer reactions (or light reactions) take place on the thylakoid membrane
2. Energy from sunlight is used to excite electrons derived from water and these high-energy electrons are carried along an electron transport chain to form NADPH and ATP; these will later be used in the dark reactions to synthesize carbohydrates from carbon dioxide
3. Photosystems are located on the thylakoid membrane
1. Photosystems are multi-protein complexes made up of two components
1. The light-harvesting complex (LHC, or antenna complex) contains proteins and pigments
2. The photochemical reaction center is a transmembrane protein-pigment complex (with a special chlorophyll a) that transfers the excited electron to a primary electron acceptor
2. The major light-absorption pigments are the chlorophylls
1. A chlorophyll molecule contains a porphyrin ring, with 4 central nitrogen (N) atoms coordinating the Mg⁺⁺ ion, plus a long hydrophobic phytol side-chain
2. Chlorophylls absorb visible light to excite electrons
3. Higher plants contain chlorophylls a and b; the two pigments complement one another as they have different light absorption spectra
4. Other accessary pigments are the carotenoids (especially b -carotene); they also absorb light energy and pass it to the chlorophylls
5. The excited electrons are converted into chemical energy in photosynthesis
4. A two-step process, known as noncyclic photophosphorylation, produces both NADPH and ATP
1. This process involves 2 photosystems, electron transport and ATP synthesis
2. The one-directional flow of electrons from water, through the two photosystems, and to NADPH is called the Z-scheme
3. At photosystem II (or P680, named after the chlorophyll a with maximum absorption at 680 nm), light is absorbed and the energy is used to split water (to generate oxygen and electrons)
4. High-energy electrons are ferried by the electron carrier plastoquinone to the cytochrome b₆-f complex, where the energy is utilized for proton pumping from the stroma to the thylakoid space
5. From the cytochrome complex, electrons are ferried by the carrier plastocyanin to the photosystem I
6. At photosystem I (or P700, due to chlorophyll a with 700 nm maximum absorption), the electrons are further energized to a very high energy level
7. The electrons are ferried to a ferrodoxin, and finally to NADP reductase to generate NADPH
8. Proton pumping at the cytochrome b₆-f complex generates an electrochemical proton gradient
1. The end result is pH 5 in the thylakoid space and pH 8 at the stroma
2. Backflow of protons via the chloroplast ATP synthase (also called CF₀CF₁ ATPase) leads to ATP synthesis
5. Cyclic photophosphorylation involves the photosystem I only and yields ATP, but no NADPH synthesis; it is so named because the electrons are transferred back to the same photosystem I
6. Experimentally, isolated chloroplasts can be shown to generate ATP in the presence of an artificially generated pH gradient
1. This experiment was reported by Andre Jagendorf in 1967
2. Isolated grana of chloroplasts were equilibrated in a pH 4 buffer
3. The pH 4-equilibrated grana were transferred into a pH 8 medium
4. In the presence of ADP and Pi, ATP was synthesized
5. This provides an experimental demonstration of the chemiosmotic hypothesis of Peter Mitchell
7. Some herbicides act by disrupting photosynthesis
1. The compound atrazine blocks the transfer of electrons out of photosystem II, and the compound paraquat accepts electrons from photosystem I
2. Selectivity is important: crops such as corn can break down the herbicide, whereas weeds cannot
3. Resistant weeds can develop by mutations in the binding sites of the target protein of the herbicide
3. The dark reactions
1. The NADPH and ATP from the light reactions are used to make carbohydrates; the reactions do not require light and are called the "dark reactions"
2. The dark reactions take place in the stroma
1. This is the carbon-fixation cycle or the Calvin cycle (also known as Calvin-Benson cycle)
2. Carbohydrates are formed from CO₂ and H₂O
3. The most important enzyme is the ribulose-1,5-bisphosphate carboxylase-oxygenase (or RuBisCO)
1. The enzyme is a multi-protein complex (550 Kd) made up of 8 large subunits and 8 small subunits
2. The large subunit is coded by a chloroplast gene, and the small subunit is coded by a nuclear gene
3. It is a slow enzyme that processes 3 substrates per sec (compared to 1000 molecules per sec for a typical enzyme)
4. The enzyme constitutes to over 50% of total chloroplast protein
5. RuBisCO is thought to be the most abundant protein on earth
6. The enzyme uses ribulose-1,5-biphosphate and CO₂ to form a transient 6 C compound (fix CO₂)
4. The products of the Calvin cycle are the 3 C compounds (triose phosphates), which are then transported to the cytosol (by translocases)
5. At the cytosol, the 3 C compounds are converted to 6 C compounds (fructose and glucose phosphates), and to the disaccharide sucrose (a form for sugar transport in plants) or to starch (storage form)
3. The dark reactions are very efficient
1. For the synthesis of 1 glucose (686 kcal/mol), it takes 12 NADPH (52 kcal/mol) and 18 ATP (7 kcal/mol) molecules
2. The efficiency is 90% ((12 x 52) + (18 X 7)) / 686 = 0.9
4. The chemical formula for photosynthesis is:

6 CO₂ + 12 H₂O -- C₆H₁₂O₆ + 6 O₂ + 6 H₂O

5. The overall efficiency of photosynthesis is about 27%, and only about 0.4% of the total sunlight energy reaching the earth is stored annually by the process of photosynthesis

1. General information
1. Chloroplasts come from immature pro-plastids
2. The formation of chloroplasts is due to coordination of both nuclear and chloroplast genes
2. Organelle genetics
1. Chloroplasts arise from pre-existing organelles
1. The forms known as proplastids are precursors to all plastids
2. Proplastids are small organelles that have relatively undifferentiated stroma, few or no thylakoids, and inner and outer membranes that surround them
3. The various forms of plastids are potentially interconvertible, depending on the growth conditions of the plant
4. In most higher plants, chloroplast genes are maternally inherited
1. In these cases, chloroplasts of the pollen grains do not enter the zygotes
2. Biparental inheritance is seen in cases where pollen chloroplasts enter the zygotes
2. The formation of the chloroplast depends on the interaction and coordination of the nuclear and the chloroplast genome
3. Nuclear-coded chloroplast proteins are made in the cytosol and transported into the chloroplast
1. Protein targeting again obeys the signal hypothesis
2. As with the nuclear gene-encoded mitochondrial proteins, N-terminal signal sequences are present; the nascent proteins are transferred in their denatured forms into the chloroplast stroma by passing the contact sites
3. The chloroplast signal sequence is found at the extreme N-terminal end
4. After this sequence is cleaved by a signal peptidase (stromal processing peptidase), the thylakoid transfer sequence is revealed
5. The protein is ferried to the thylakoid lumen, where another signal peptidase, thylakoid processing peptidase, cleaves the signal and the protein is folded
6. Chaperone proteins help in protein folding; thus, for RuBisCO, the chloroplast-encoded large subunit is prevented from aggregate formation by a binding protein (chaperonin) until the nucleus-encoded small subunit can be ferried into the stroma from the cytosol
4. A chloroplast has its own genetic system
1. There are 50-100 copies of DNA per chloroplast, and chloroplast DNA constitutes up to 6% of total DNA of a plant cell
2. Chloroplast DNAs are circular molecules that are 120-160 Kb in size; each codes for about 120 genes
3. The complete chloroplast genome has been sequenced
1. The chloroplast DNA of liverwort Marchantia polymorpha has 121,024 base pairs, the dicot tobacco Nicotiana tabacum, 155,844 base pairs, and the monocot rice Oryza sativa, 134,525 base pairs
2. Circular DNA can be divided into 4 regions: a large single copy region and a small single copy region that are separated by 2 inverted repeats
4. The chloroplast DNA-encoded genes include 4 rRNAs, about 20 ribosomal proteins, 30 tRNAs, and proteins (such as subunits for RNA polymerase, for photosystems I and II, for ATP synthase, and about 40 proteins of unknown function)
5. Some proteins are unidentified (ORF or open-reading frames) and some proteins come from genes containing introns
6. Chloroplast DNA-encoded proteins have characteristics of prokaryotic protein synthesis
1. Chloroplast ribosomes resemble bacterial ribosomes in some respects; for example, both are sensitive to the antibiotics chloramphenicol and tetracycline
2. Protein synthesis in chloroplasts starts with N-formyl Met, as in bacteria (not Met, as found in eukaryotes)
3. Chloroplast DNA can be transcribed by bacterial RNA polymerase; chloroplast mRNA can be translated in a bacterial system
4. These results support the endosymbiont origin of the chloroplast

1. Give examples of the different proteins and enzymes found in the various compartments of the chloroplast.
2. Describe the function of photosystems I and II and RuBisCO in photosynthesis.
3. Trace the targeting steps by which a nucleus-coded chloroplast protein reaches the thylakoid membrane.
4. List the various arguments that favor the endosymbiont origin of the chloroplast.