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CARBON FIXATION

CARBON FIXATION. THE LIGHT INDEPENDENT REACTION. THE LIGHT INDEPENDENT REACTION. Enzyme controlled (Q 10  2) Located in the stroma of the chloroplast Occurs simultaneously with the light dependent reaction

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CARBON FIXATION

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  1. CARBON FIXATION THE LIGHT INDEPENDENT REACTION

  2. THE LIGHT INDEPENDENT REACTION • Enzyme controlled (Q10 2) • Located in the stroma of the chloroplast • Occurs simultaneously with the light dependent reaction • It can continue in the dark provided the necessary raw materials are available (CO2, NADPH + H+ and ATP) © 2010 Paul Billiet ODWS

  3. Enzyme controlled reaction pathways A B C D E F Ee Ea Ec Ed Ea X = Precursor (raw material) added here © 2010 Paul Billiet ODWS

  4. Enzyme controlled reaction pathways To find out the sequence of the reactions and the point at which Xis added in, two approaches can be used: • Label and trace the products formed through time • Cut the supply of X and observe what happens to the intermediates in the pathway e.g. in studying photosynthesis, cut the CO2 supply or switch off the lightso cutting the supply of ATP and NADPH+H+ © 2010 Paul Billiet ODWS

  5. Image Credit Scenedesmus Calvin and Benson 1946 to 1953 • Used 14C radioisotope for labelling • Unicellular algae: Chlorella and Scenedesmus • Simple plants which respond quickly to changes in the environment • So little time lag © 2010 Paul Billiet ODWS

  6. The “Lollipop” vessel A flat-sided, round flask containing the culture of algae This shape:- provided even illumination of all the cells- permitted careful control of environmental conditions (e.g. pH, temperature)- permitted rapid mixing of contents- precise sampling time © 2010 Paul Billiet ODWS

  7. Labelling and tracing carbon using 14C • Add NaH14CO3 solution • At timed intervals the algae are sampled and killed by dropping in hot methanol • Two-way (2-dimensional) chromatography used to separate the compounds • Identify radioactively labelled compounds by autoradiography © 2010 Paul Billiet ODWS

  8. A. Mixture placed at the origin C. Rotate the paper 90° B.1st run E. Autoradiograph reveals the compound/s which are labelled with 14C D. 2nd run Two-way (2-dimensional) chromatography © 2010 Paul Billiet ODWS

  9. 2s Results Salisbury & Ross (1969) Plant Physiology Wadsworth

  10. Result • The compound that became labelled first was Phosphoglyceric Acid (or Phosphoglycerate) PGA • PGA is a 3 carbon sugar • Therefore: Compound B + CO2 PGA What is compound B?!!! © 2010 Paul Billiet ODWS

  11. Light independent pathway A B PGA D E F Ee Ea Ec Ed Ea CO2 What is compound B?!!! © 2010 Paul Billiet ODWS

  12. Searching for the compound that combines with carbon dioxide • Feed the algae NaH14CO3 for over 15 min • All the compounds involved in photosynthesis are labelled • Then reduce the level of NaH14CO3 rapidly Prediction • PGA levels should fall rapidly • The compound that fixes CO2 should rise sharply © 2010 Paul Billiet ODWS

  13. Salisbury & Ross (1969) Plant Physiology Wadsworth

  14. Result • This compound was found to be Ribulose Bisphosphate (RBP) • RBP is a 5 carbon sugar Therefore RBP + CO2 2PGA 5C + 1C = 2x3C • The enzyme controlling this important step is Ribulose Bisphosphate Carboxylase (Rubisco) © 2010 Paul Billiet ODWS

  15. Light independent pathway A RBPPGA D E F Ee RUBISCO Ec Ed Ea CO2 © 2010 Paul Billiet ODWS

  16. RUBISCO A large enzyme 500 000 Daltons Inhibited by oxygen A relatively slow enzyme 50% of chloroplast protein Probably the most abundant protein on Earth Consists of two subunits (1 large + 1 small) The large subunit is coded for by a gene on chlDNA Image Credit: Rubisco stick model © 2010 Paul Billiet ODWS

  17. Carbon fixation and light • The light dependent pathways produce ATP and NADPH+H+ to be used in the light independent processes • Illuminate the algae for 15 min then cut the light Result • PGA levels rise • Other compounds, after PGA in the pathway, fall © 2010 Paul Billiet ODWS

  18. Carbon fixation and light • The compound requiring the NADPH+H+ and ATP was found to be Glycerate 3-phosphate (GP) • GP is converted to various other three carbon sugars or Triose Phosphates (TP) some are used to produce hexoses such as fructose phosphate and glucose phosphate © 2010 Paul Billiet ODWS

  19. Light independent pathway A RBPPGAGP E Hexoses Ee RUBISCO Ec Ed Ea 12 ATP 12 NADPH + H+ CO2 © 2010 Paul Billiet ODWS

  20. Image Credit: Salisbury & Ross (1969) Plant Physiology Wadsworth A curious turn • When the light is cut interestingly RBP levels also fall • This is for two reasons • RBP requires ATP in its synthesis • RBP is formed from some of the GP • In other words the reaction pathway is a CYCLE © 2010 Paul Billiet ODWS

  21. Squaring the circle! The Calvin-Benson Cycle 6 RBP (5C) 6CO2 (1C) 6 ATP 12 PGA (3C) Intermediates 12 TP (3C) 12 GP (3C) 1 Hexose (6C) 12 ATP 12 NADPH + H+

  22. CHLOROPLAST CYTOSOL Light dependent reaction O2 H2O ATP NADPH+H+ Light independent reaction CO2 Sugars Triose phospates Fattyacids Amino acids Starch PHOTOSYNTHESIS OVERVIEW © 2010 Paul Billiet ODWS

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