1. CARBON FIXATION THE LIGHT INDEPENDENT REACTION

2. Enzyme controlled (Q 10  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 (CO 2, NADPH + H + and ATP) © 2010 Paul Billiet ODWSODWS

3. ABCDEFABCDEF Enzyme controlled reaction pathways Ea EcEd Ee X = Precursor (raw material) added here © 2010 Paul Billiet ODWSODWS

4. Enzyme controlled reaction pathways To find out the sequence of the reactions and the point at which X is added in, two approaches can be used: 1. Label and trace the products formed through time 2. Cut the supply of X and observe what happens to the intermediates in the pathway e.g. in studying photosynthesis, cut the CO 2 supply or switch off the light so cutting the supply of ATP and NADPH+H + © 2010 Paul Billiet ODWSODWS

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

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 ODWSODWS

7. Labelling and tracing carbon using 14 C Add NaH 14 CO 3 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 ODWSODWS

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

9. Results 2s 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 + CO 2  PGA What is compound B?!!! © 2010 Paul Billiet ODWSODWS

11. ABPGADEF Light independent pathway Ea EcEd Ee CO 2 What is compound B?!!! © 2010 Paul Billiet ODWSODWS

12. Searching for the compound that combines with carbon dioxide Feed the algae NaH 14 CO 3 for over 15 min All the compounds involved in photosynthesis are labelled Then reduce the level of NaH 14 CO 3 rapidly Prediction PGA levels should fall rapidly The compound that fixes CO 2 should rise sharply © 2010 Paul Billiet ODWSODWS

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 + CO 2  2PGA 5C + 1C = 2x3C The enzyme controlling this important step is Ribulose Bisphosphate Carboxylase (Rubisco) © 2010 Paul Billiet ODWSODWS

15. ARBPPGADEF Light independent pathway Ea RUBISCO EcEd Ee CO 2 © 2010 Paul Billiet ODWSODWS

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 Rubisco stick model © 2010 Paul Billiet ODWSODWS

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 ODWSODWS

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 ODWSODWS

19. ARBPPGAGPEHexoses Light independent pathway Ea RUBISCO EcEd Ee CO 2 12 ATP 12 NADPH + H + © 2010 Paul Billiet ODWSODWS

20. 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 Image Credit: Salisbury & Ross (1969) Plant Physiology Wadsworth © 2010 Paul Billiet ODWSODWS

21. Squaring the circle! The Calvin-Benson Cycle 6 ATP 6CO 2 (1C) 6 RBP (5C) 12 PGA (3C) 12 ATP 12 NADPH + H + 12 GP (3C) 12 TP (3C) 1 Hexose (6C) Intermediates

22. PHOTOSYNTHESIS OVERVIEW CHLOROPLASTCYTOSOL H2OH2O O2O2 Light dependent reaction Light independent reaction CO 2 Starch Sugars Fatty acids Amino acids ATPNADPH+H + Triose phospates © 2010 Paul Billiet ODWSODWS