BIOL231 Ch 14 FA2024-2 PDF

CHAPTER 14 Energy Generation in Mitochondria Complete Oxidation of Glucose Yields: Pathway ATP NADH FADH2 CO2 Glycolysis 2 2 0 0 PDH 0 2 0 2 Kreb 2 6 2 4 Total 4 10 2 6  Ce...

CHAPTER 14 Energy Generation in Mitochondria Complete Oxidation of Glucose Yields: Pathway ATP NADH FADH2 CO2 Glycolysis 2 2 0 0 PDH 0 2 0 2 Kreb 2 6 2 4 Total 4 10 2 6  Cellular Respiration Electrons (from high E electron carriers) flow through a series of membrane-bound electron carriers to a final electron acceptor generating ATP Cellular Respiration is a Two Step Process 1) Electron Transport Chain - Energy Oxidative transfers from high energy electron Phosphorylation carriers to form a proton gradient 2) ATP Synthase - Proton gradient drives ATP (FADH2) (FAD) synthesis Occurs in the membranes of Proton Gradient mitochondria in Eukaryotes  Does this mean that prokaryotes can’t undergo cellular respiration? ***Recall the endosymbiotic theory Structure of the Mitochondria Outer Membrane Permeable – porins Smooth Inner Membrane Impermeable Folded - Cristae Intermembrane space matrix  Pathways are found in Specific Locations within the Mitochondria  Matrix  Pyruvate Dehydrogenase Complex  Krebs Cycle  mtDNA/ribosomes  Fatty acid catabolism  Inner membrane - 75% protein by weight  Electron Transport Chain Proteins are  ATP synthase embedded in the membrane Oxidative Phosphorylation involves both 1) Electron Transport Chain (respiratory chain) 2) ATP synthase Functionally linked to each other Overview: Electron Transport Chain (ETC) Electrons donated to ETC from NADH and FADH2 As e- flow through ETC, protons are moved from matrix to intermembrane space Forms a proton gradient – form of potential energy Five Types of Electron Carriers found in the ETC Complexes Most are hydrophobic - suspended in inner membrane Most contain prosthetic groups that undergo oxidative/reduction reactions Five Types of Electron Carriers found in the ETC Complexes FLAVIN 1. Flavoproteins – contain FAD or FMN 2. Iron-sulfur proteins –contain Iron-sulfur centers  Five Types of Electron Carriers found in the ETC Complexes ex. Cytochrome C 3. Cytochromes - proteins that Peripheral contain heme groups (Fe membrane bound containing porphyrin ring) protein on the side of the Intermembrane space 4. Copper-containing cytochromes (Cu associated with heme group) Five Types of Electron Carriers found in the ETC Complexes 5. Coenzyme Q – only nonprotein component of ETC Coenzyme Q Very lipid soluble Get to know your ETC Complexes!!! Overview ETC = series of proteins in inner mitochondrial membrane … Outer membrane Intermembrane space Inner membrane Overview NADH, FADH2 deliver e– and H+ to ETC e– moves from protein to protein, then to O2 Outer membrane Intermembrane space Inner membrane  Intermembrane space Succinate Cytochrome dehydrogenase c reductase Cytochrome c Ubiquinone (Q) NADH Cytochrome c dehydrogenase oxidase e e Inner membrane e e e 2 FADH e NADH + H+ H+ ½ O2 + 2H+ H2O Matrix  Intermembrane space H+ H+ H+ Cytochrome H+ c reductase H+ H+ NADH Cytochrome c dehydrogenase oxidase Inner membrane H+ H+ H+ H+ H+ H+ Matrix Function of Electron Transport Chain is two-fold Generates a Proton Gradient Regenerates FAD and NAD+ Electron transport is highly Exergonic Don’t memorize values! Why are electrons passed from one molecule to another? Why not directly to oxygen? As electrons flow through the ETC, protons move from the matrix to the intermembrane space High Conc. of protons [H+] decreases in matrix (pH?) Low [H+] More negative membrane potential We are creating an electrochemical gradient by the unequal distribution of protons  Intermembrane space Succinate Cytochrome dehydrogenase c reductase Cytochrome c Ubiquinone (Q) NADH Cytochrome c dehydrogenase oxidase e e Inner membrane e e e 2 FADH e NADH + H+ H+ ½ O2 + 2H+ H2O Matrix Structure of ATP Synthase Outer Cytosol Mitochondrial membrane Inner mitochondrial membrane Intermembrane space – low pH Matrix – higher pH Electron Transport Chain The higher negative charge in the matrix attracts the protons (H+) back from the intermembrane space to the matrix. The accumulation of protons in the intermembrane space drives protons into the matrix via diffusion. We have a Proton Dam! Potential Energy! H+ H+ H+ H+ H+ H+ + + H+ H+ HH H+H+ H+ H+ Matrix How do we capture this energy? H++ Cells do this using H HH + the ATP Synthase! + H+ H+ H++ HH+ Structure of the ATP Synthase ATP Synthase F0 - pore or opening/ turns like a rotor F1 – ATPase activity Structure of ATP Synthase  ATP Synthase H+ flow through ATP Synthase (intermembrane space back to matrix) Energy of electrochemical gradient drives ATP synthesis ATP Synthase – more specifically…… ADP + Pi binds to F1 subunits Flow of protons through the F0 complex into the matrix forces rotor to turn Rotor turns forcing conformational change in F1 subunits Phosphoanhydride bond forms between ADP + Pi making ATP (E is stored!)  Intermembrane space H+ H+ H+ H+ H+ H+ ATP synthase Rotor/F0 unit Stalk/stator Catalytic head/F1 unit ADP+Pi ATP H+ WATCH THIS ANIMATION! https://vimeo.com/garlandscience3 0308032/review/189355245/563c5 Matrix 967ee Draw a picture of the ETC and the ATP synthase in the mitochondria. What do you Label the complexes Place them in the correct remember? location. Label your mitochondrial membranes and spaces REMINDER: Get to know your ETC Complexes!!! ATP synthase has two major functions: (1) to provide a channel through which H+ can flow (chemiosmosis) (2) to use the energy of that flow to phosphorylate ADP to ATP (oxidative phosphorylation)  Putting it all together…. NADH donates its electrons to Complex I/FADH2 to Complex II Electrons flow through the ETC to oxygen to form water Protons flow back to the intermembrane space into the matrix through the ATP synthase Protons are moved from the matrix to the ATP synthase uses potential intermembrane space energy to couple ADP + Pi to form ATP  How much ATP can be generated? Theoretically…… Two electrons from NADH Two electrons from FADH2 contribute to the proton contribute to the proton gradient to provide energy to gradient to provide energy to synthesize 3 moles of ATP synthesize 2 moles of ATP NADH NAD+ FADH2 FAD  Why does one NADH yield three ATP, but one FADH2 only yields two? NADH feeds its electrons into the electron transport chain at the beginning (NADH dehydrogenase). FADH2 feeds into the electron transport chain at succinate dehydrogenase complex (at a lower energy level down the chain). The high energy electrons from NADH have sufficient energy to result in 3 ATP whereas the lower energy electrons in FADH2 have only energy for 2 ATP. How many Moles of ATP are generated from One Mole of Glucose in Aerobic Conditions? Pathway ATP NADH FADH2 CO2 Glycolysis 2 2 0 0 PDH 0 2 0 2 Krebs 2 6 2 4 Total 4 10 2 6  Theoretically, how many Moles of ATP are generated from One Mole of Glucose in Aerobic Conditions? ATP NADH FADH2 Total 4 10 2 X3 X2 4 + 30 + 4 = 38 mole of ATP/mol of glucose  How efficient is cellular respiration in capturing the energy released in one mole of glucose as ATP? 1 Glucose = -686 kcal/mol 1 ATP = -7.3 kcal/mol Amt of Energy captured as ATP 38 ATP * -7.3 kcal/mol = 277.4 kcal/mol X 100 = 40.4% Amt of Energy original stored in glucose X 100 = -686 kcal/mol in glucose Efficient Energy Yield of Respiration theoretical energy yields - 38 ATP per glucose for bacteria - 36 ATP per glucose for eukaryotes….why the difference? (think about glycolysis) actual energy yield - ~29 ATP per glucose for eukaryotes - reduced yield is due to “leaky” inner membrane (leaky to H+); and due to active transport of pyruvate, ADP and Pi into the mitochondria  Respiration Overview ox. C6H12O6 + 6O2 6CO2 + 6H2O + E red. What is the major limiting factor of aerobic respiration? O2 What happens to Energy Production in the Absence of Oxygen? ETC? ATP Synthase? Kreb Cycle? PDH? Glycolysis?  In the presence of oxygen, all is good! But if no oxygen…… Glucose 2ADP + Pi X XXX FADH2 2 ATP X X Acetyl CoA 2 NAD+ PDH X 2 2 Pyruvate NADH Oxidation Without O2 Respiration occurs without O2 via two ways: 1. anaerobic respiration -use of inorganic molecules (other than O2) as final electron acceptor 2. Fermentation -use of organic molecules as final electron acceptor so glycolysis continue to provide ATP  Oxygen is Final Electron Acceptor in Aerobic Respiration Anaerobic Respiration sulfur, protons, ferric ions are examples Final Electron acceptors (Anaerobic bacteria) Oxidation Without O2 Anaerobic respiration by methanogens: methanogens use CO2 CO2 is reduced to CH4 (methane) Anaerobic respiration by nitrate reduction (in some bacteria, like E. coli): N03- + 2e- + 2H+  N02-+ H20 The reaction for nitrate reduction. N03-, nitrate; N02-, nitrite Anaerobic respiration by sulfur bacteria inorganic sulphate (SO4) is reduced to hydrogen sulfide (H2S) Oxidation Without O2 Respiration occurs without O2 via two ways: 1. anaerobic respiration -use of inorganic molecules (other than O2) as final electron acceptor 2. Fermentation -use of organic molecules as final electron acceptor so glycolysis continue to provide ATP  In fermentation, we want to find a way for glycolysis to function! But if no oxygen…… Glucose 2ADP + Pi X XXX FADH2 2 ATP X X Acetyl CoA 2 NAD+ PDH X 2 2 Pyruvate NADH  Fermentation allows Glycolysis to Function in the Absence of Oxygen Anaerobic conditions – no Oxygen Fermentation Glucose Allows glycolysis to continue to 2ADP + Pi produce small amounts of ATP Uses pyruvate to recycle NADH to 2 ATP NAD+ 2 NAD+ Two Types of Fermentation Lactic Acid Fermentation 2 NADH Alcohol Fermentation 2 Pyruvate Oxidation Without O2 Fermentation reduces organic molecules in order to regenerate NAD+ so glycolysis can continue 1. ethanol fermentation occurs in yeast -CO2, ethanol, and NAD+ are produced 2. lactic acid fermentation -occurs in animal cells (especially muscles) and some bacteria -electrons are transferred from NADH to pyruvate to produce lactic acid Alcohol Fermentation What are the net products of alcohol fermentation? Where does it take place in ADH the cell? Lactic Acid Fermentation What are the net products of lactic acid fermentation? Where does it take place in the cell? LDH How much energy is captured as ATP from glucose during fermentation? 1 Mole Glucose = -686 kcal/mole 1 Mole ATP = -7.3 kcal/mol ( ) 2 ATP X -7.3 kcal/mol -686 kcal/mol X 100 = 2.13 % a pittance, but better than nothing!!!  Other Sources of Energy: Poly- and Disaccharides Disaccharides monosaccharides Polysaccharides glucose *or* glycolytic intermediates  Other Sources of Energy: Fats Fats (TAGs) 3 Fatty Acids + Glycerol β-oxidation Acetyl CoA Krebs Cycle Glycolytic intermediate  Catabolism of Fat Catabolism of fats: -fats are broken down to fatty acids and glycerol -fatty acids are converted to acetyl groups by β-oxidation The respiration of a 6-carbon fatty acid yields 20% more energy than glucose. WHY?  Other Sources of Energy: Protein Protein Amino Acids Deamination to remove nitrogen Acetyl CoA, Glycolytic or Kreb Cycle intermediates  Catabolism of Protein Catabolism of proteins: -amino acids undergo deamination to remove the amino group; also transamination reactions -remainder of the amino acid is converted to a molecule that enters glycolysis or the Krebs cycle -for example: alanine is converted to pyruvate aspartate is converted to oxaloacetate Regulation of Respiration Regulation of aerobic respiration is by feedback inhibition: - high levels of ATP and by citrate inhibit phosphofructokinase - high levels of NADH inhibit pyruvate dehydrogenase - high levels of ATP inhibit citrate synthetase Chapter 14 Part II: PHOTOSYNTHESIS  Photosynthesis Overview ox. 6CO2 + 12H2O C6H12O6 + 6H2O + 6O2 red. red. ox. Photosynthesis is divided into: light-dependent reactions (“photo”) capture energy from sunlight make ATP and reduce NADP+ to NADPH O2 generated carbon fixation reactions /Calvin cycle (“synthesis”) use ATP and NADPH to synthesize organic molecules from CO2  If we focus on plants… Cuticle Epidermis Mesophyll Vascular bundle Bundle Stoma sheath  Outer Cuticle Inner Thylakoids membrane membrane Upper epidermis (palisade) mesophyll Bundle sheath Vein Xylem Phloem (spongy) mesophyll Lower epidermis Intermembrane space Thylakoid Stoma Guard cells Thylakoid Stroma Granum lumen (a) Leaf cross section membrane (stack of thylakoids) (c) Chloroplast cross section  Structure of a Chloroplast Outer membrane (porins) Inner membrane Thylakoid membrane Thylakoids Granum Stromal thylakoids Intermembrane space Stroma (contains DNA and ribosomes) Thylakoid Lumen Similarities with the mitochondria? But realize they are also very different! Pigments Pigments: molecules that can absorb different wavelengths of light Main: chlorophyll a accessory pigments: secondary pigments absorbing light wavelengths other than those absorbed by primary pigment molecules (665 nm and 465 nm) chlorophyll b carotenoids Xanthophyll carotene anthocyanins -increase the range of light wavelengths that can be used in photosynthesis  Light Rxns Solar E to Chemical E Pigments – light absorbing molecules photons = discrete packets of Energy  Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Chlorophyll molecules embedded in a H2C CH H R protein complex in the thylakoid CH3 CH2CH3 Porphyrin N N membrane Mg H head H N N Thylakoid CH3 CH3 membrane H H CH2H CH2 O O C CO2CH3 O CH2 CH CCH3 CH2 CH2 Thylakoid CH2 CHCH Hydrocarbon CH2 tail CH2 CH2 CHCH3 CH2 CH2 CH CHCH3 Granum CH3 Chlorophyll a: R — CH3 Chlorophyll b: R —CHO What happens when a pigment absorbs a photon (3 possibilities)? Electron can go back to ground Excited State state by (High Energy Photon of and unstable) Light 1. Releasing energy as heat/fluorescence e- 2. Passing exited energy to a Ground State nearby pigment (low Energy) – But what happens to the pigment What the pigment does with the excited that gains the energy? electron is dependent on its role in photosynthesis. 3. Pass excited electron to an ETC  Photon Photon is absorbed by an excitable electron that moves into a higher energy level. Low energy level High energy level Electron Either Or Electron acceptor molecule The electron may return The electron may be to ground level by emitting accepted by an electron a less energetic photon. acceptor molecule.  Photosystem Organization A photosystem consists of 1. an antenna complex of hundreds of accessory pigment molecules 2. a reaction center of a “special pair” chlorophyll a molecules (hold their electrons at a lower energy then other chlorophylls) Energy of electrons is transferred through the antenna complex to the reaction center.  Thylakoid Primary electron acceptor Chloroplast Reactor center Proton Photosystem Antenna complexes Photosystem Organization (summary) At the reaction center, the energy from the antenna complex is transferred to chlorophyll a. This energy causes an electron from chlorophyll to become excited. The excited electron is transferred from chlorophyll a to an electron acceptor. Water donates an electron to chlorophyll a to replace the excited electron (photolysis).  The Production of ATP… Photophosphorylation---synthesis of ATP coupled to the transport of electrons that have been energized by photons of light We will focus on plants, where two linked photosystems are used in noncyclic photophosphorylation 1. photosystem I -reaction center pigment (P700) with a peak absorption at 700nm 2. photosystem II -reaction center pigment (P680) has a peak absorption at 680nm  Photosystems I and II PSII – P680 Names of special chlorophyll molecules in reaction centers based on wavelength of PSI - P700 max absorption Photosystem II When excited, an electron from P680 passes to ETC As electrons pass down ETC, protons are pumped across the thylakoid membrane from the stroma into the thylakoid lumen (space) An ATP synthase uses proton gradient to generate ATP as protons flow back from thylakoid lumen to stroma Final Electron Acceptor is P700 (Reaction Center of PSI) Light-Dependent Reactions Photosystem II acts first: -accessory pigments shuttle energy to the P680 reaction center -excited electrons from P680 are transferred to b6-f complex, via intermediates -electron lost from P680 is replaced by an electron released from the splitting of water Light-Dependent Reactions The b6-f complex is a series of electron carriers. -electron carrier molecules are embedded in the thylakoid membrane -protons are pumped into the thylakoid space to form a proton gradient  Pheophytin Step 1: Photon strikes PS II Energy relayed to P680  excited Step 2: e– transferred to pheophytin P680 Enzyme splits H2O … lost e– replaced O2 formed ! Photosystem II Step 3: e– passes through 1st ETC Energy drop used to make ATP Plastoquinone Cytochrome complex (b6-f) Plastocyanin Photosystem II Light-Dependent Reactions Photosystem I -receives energy from an antenna complex -energy is shuttled to P700 reaction center -excited electron is transferred to a membrane-bound electron carrier -electrons are used to reduce NADP+ to NADPH -electrons lost from P700 are replaced from the b6-f complex Photosystem I Figure 14-37 When excited, an electron from P700 passes to ETC Electrons flow through ETC Final electron acceptor is NADP+ forming NADPH Electron of P700 is replaced from PSII NO PROTON GRADIENT FORMED NO OXYGEN IS GENERATED  Step 4: 2 photons strike PS I Energy relayed to P700  excited Ferrodoxin Ferredoxin NADP reductase (FNR) NADP+ NADPH P700 Photosystem I Photosystem II Step 5: e– passes through 2nd ETC Enzyme reduces NADP+ Putting it all Together… Light-Dependent Reactions ATP is produced via chemiosmosis. - ATP synthase is embedded in the thylakoid membrane -protons have accumulated in the thylakoid space -protons move into the stroma only through ATP synthase -ATP is produced from ADP + Pi  Stroma Thylakoid lumen Thylakoid membrane Protons (H+) Putting it all Together… P680 P700 Products of the Energy Transduction Reactions Photosystem II Generates a proton gradient used to synthesize ATP by an We now have ATP synthase captured solar Oxygen released energy into the form of ATP and Photosystem I NADPH! NADPH is generated  Dark Reactions (Carbon Fixation or Calvin Cycle) Occurs in the Stroma Synthesis of organic molecules from carbon dioxide Uses ATP and NADPH made in the light dependent reactions Fix carbon dioxide into a 3 Carbon sugar – Glyceraldehyde 3- Phosphate Calvin Cycle  Reaction Catalyzed by Ribulose 1,5 Bisphosphate Carboxylase/Oxygenase (RUBISCO) (1C) (5C) (6C) (2x 3C) 40 million tons of Rubisco on earth!! Calvin Cycle Rubisco 1. Carbon fixation 3 x CO2 Rubisco 3 x Ribulose 1,5-bisphosphate 6x 3-phosphoglycerate 2. Carbon reduction 3 x CO2 Rubisco 3 x Ribulose 1,5-bisphosphate 6x 3-phosphoglycerate 6 ATP PGK 6 ADP 6 x 1,3-bisphosphoglycerate 5x G3P 6 NADPH 6Pi G3P DH 6 NADP+ 6x Glyceraldehyde 3-phosphate G3P Sugars 3. RuBP regeneration 3 x CO2 Rubisco 3 x Ribulose 1,5-bisphosphate 6x 3-phosphoglycerate 3 ADP 2Pi PRK 3 ATP 6 x 1,3-bisphosphoglycerate 5x G3P 6x Glyceraldehyde 3-phosphate G3P Sugars  Calvin Cycle Overall CC rxn: 3 CO2 + 6 NADPH + 6 H+ + 9 ATP → G3P + 6 NADP+ + 9 ADP + 3 H2O + 8 Pi Requires 9 ATP and 6 NADPH generated in the light dependent reactions to form one molecule of glyceraldehyde 3-phosphate from 3 molecules of CO2 RECALL from LDR: 6H2O + 6NADP+ + 9ADP + 9Pi → 3O2 + 6NADPH + 9ATP  Fate of Glyceraldehyde 3-Phosphate Converted to glucose in the stroma of the chloroplast stored as starch Released into cytoplasm and converted to glucose and fructose Used to make sucrose – transported to rest of plant Both starch and sucrose can be broken down for energy when needed– can enter glycolysis/cellular respiration for energy Calvin Cycle Carbon Fixation Reactions The energy cycle: -photosynthesis uses the products of respiration as starting substrates -respiration uses the products of photosynthesis as starting substrates The relationship is complementary!!!  BIOL231 FA22 Exam III (n=57, avg. 74.3%) 16 14 12 10 8 6 4 2 0 0-50 50-60 60-70 70-80 80-90 90-100 Major limiting factors in photosynthesis: Light Temperature CO2 What happens if CO2 levels get too low??? Photorespiration Rubisco has 2 enzymatic activities: 1. (carboxylase) carboxylation – the addition of CO2 to RuBP -favored under normal conditions; we saw this with C3 cycle 2. (oxygenase) photorespiration – the oxidation of RuBP by the addition of O2 -favored in hot conditions CO2 and O2 compete for the active site on Rubisco. Photorespiration Occurs in the presence of light Requires O2 like aerobic respiration Produces CO2, H2O like aerobic respiration BUT, no ATP is produced, thus decreasing the photosynthetic efficiency of the plant, because it uses up the intermediates of the Calvin cycle  Photorespiration occurs when the CO2 levels inside a leaf become low. This happens on hot, dry days when a plant is forced to close its stomata to prevent excess water loss. If the plant continues to attempt to fix CO2 when its stomata are closed, the CO2 will get used up and the O2 ratio in the leaf will increase relative to CO2 concentrations. When the CO2 levels inside the leaf drop to around 50 ppm, Rubisco starts to combine O2 with RuBP instead of CO2. The net result of this is that instead of producing 2 3C PGA molecules, only one molecule of PGA is produced and a toxic 2C molecule called phosphoglycolate is produced.  Photorespiration The plant must get rid of the phosphoglycolate First it immediately gets rid of the phosphate group, converting the molecule to glycolic acid. The glycolic acid is then transported to the peroxisome and there converted to glycine. The glycine is then transported into a mitochondria where it is converted into serine. The serine is then used to make other organic molecules. All these conversions cost the plant energy and results in the net loss of CO2 from the plant. Photosynthetic adaptations Some plants can avoid photorespiration by using an enzyme other than RUBISCO. -PEP carboxylase -CO2 is added to phosphoenolpyruvate (PEP) -a 4 carbon compound is produced -CO2 is later released from this 4-carbon compound and used by rubisco in the Calvin cycle Photosynthetic adaptations C4 plants (angiosperms/flowering plants, corn, sugarcane, amaranth) - use PEP carboxylase to capture CO2 - CO2 is added to PEP in one cell type (mesophyll cell) - the resulting 4-carbon compound is moved into a bundle sheath cell where the CO2 is released and used in the Calvin cycle  Upper epidermis Palisade mesophyll Bundle sheath cells of veins (a) Arrangement of cells in a C3 leaf (b) Arrangement of cells in a C4 leaf  CO2 Mesophyll cell Phosphoenol- (3C) Oxaloacetate (4C) pyruvate ADP NADPH ATP NADP+ Pyruvate Malate (4C) (3C) ADP (3C) Pyruvate Malate (4C) Bundle NADP+ sheath cell CO2 Glucose NADPH Vein Photosynthetic adaptations CAM (Crassulaceae family) plants crassulacean acid metabolism (orchids, cacti, pineapples) -CO2 is captured at night when stomata are open -PEP carboxylase adds CO2 to PEP to produce a 4 carbon compound -this compound releases CO2 during the day -CO2 is then used by rubisco in the Calvin cycle  Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CO2 Phosphoenol- Oxalo- pyruvate (PEP) acetate PPi + AMP Mesophyll Pi + cell CO2 CO2 ATP Pyruvate Malate Mesophyll C4 C4 cell pathway pathway Night Pyruvate Malate Mesophyll CO2 CO2 cell Bundle- CO2 sheath Bundle- Calvin Calvin Day cell sheath cycle Calvin cycle cell cycle Glucose Glucose Glucose C4 plants CAM plants C4 pathways C4 versus CAM pathways