1. Photosynthesis: the light- independent reactions Biol 3470 Plant Physiol Biotech 5.5 to 5.12 Lecture 9 Thurs. Feb. 2, 2006 From Rost et al., “Plant Biology”, 2 nd edn

2. The process of carbon fixation in plants goes by many names Including: –The dark reactions –The enzymatic reactions of photosynthesis –Reductive pentose phosphate cycle –C3 cycle –The photosynthetic carbon reduction (PCR) cycle (in the textbook)

3. The PCR cycle converts atmospheric carbon to organic molecules Convert CO 2 to stable phosphorylated carbon intermediates (specifically- a three-carbon carbohydrate, 3-PGA) Uses the energy produced in the light-dependent reactions to reduce CO 2 –Convert less complex → more complex molecules –Fight entropy Pathway elicited in late 1940s and early 50s by U.S. plant physiologist Melvin Calvin using labeled 14 CO 2 –feed plants 14 CO 2 –Allow metabolism –Kill, extract, examine small carbohydrates that contain 14 C using paper chromatography and autoradiography

4. The PCR cycle contains 3 distinct segments Step 1: Carboxylation fixes CO 2 using the enzyme rubisco 14 CO 2 fixed first into 3- phosphoglycerate (3 Cs ≡ C3 cycle) –3-PGA is the first organic product of the PCR cycle Given this product and reactant, one would assume the plant substrate of the PCR cycle would have ___Cs 1 32 Actually, the plant substrate for the PCR cycle is a five-carbon substrate, RuBP Fig. 5.8 Fig. 5.9 Unstable intermediate is hydrolyzed (2x ) Rubisco reaction

5. The key enzyme regulating carbon uptake by the PCR cycle is rubisco Rubisco Enzyme with a high affinity for CO2 Present in high amounts in the chloroplast stroma Its activity maintains a CO2 gradient from the atmosphere ΔGº′ = -35 kJ/mol (energetically favourable to occur spontaneously) But its activity requires ATP + NADPH made in the light reactions elsewhere in PCR cycle e.g. in 2 nd step: reduction of 3- PGA to G3P This is Step 2: Reduction Fig. 5.10 Phosphorylate!Reduce!

6. The final step in the PCR cycle regenerates the rubisco substrate This is accomplished via Step 3: Regeneration –Requires 1 ATP per CO2 Note that the PCR cycle is autocatalytic –This means that it operates more quickly if CO2 and/or RuBP pools are low (e.g. in the morning, when the RuBP supply is depleted)

7. PCR cycle activity must be integrated with plant carbon metabolism as a whole These include respiration (glycolysis) and macromolecule synthesis (for nucleic acids, lipids, carbohydrates, proteins) Thus, the PCR cycle activity must be regulated by a number of mechanisms The plant wants to keep CO2 fixation rate high to make more organic carbon Fig. 5.11 Rubisco Autocatalytic RuBP regeneration 2. Reduction 3. Regener- ation 1. Carbox- ylation The carbon from 5 of every 6 molecules of G3P needs to be recycled to make RuBP and keep the cycle spinning Only around one- sixth of the carbon fixed is exported from the leaf and supports growth and metabolism To keep high CO2 fixation, the plant can prevent G3P export The PCR cycle consumes the ATP and NADPH produced in the light-dependent reactions

8. Mechanisms of regulation of PCR cycle activity The activity of rubisco is regulated by light Complex mechanism driven by uptake of protons by thylakoid lumen between 1.Mg 2+ → moves lumen → stroma to compensate for H + uptake by thylakoids in light inactiveactive (light) Dark stroma pH = 5.0 Light Fig. 5.14 Stromal pH ↑ activates rubisco 2.CO 2 → binds to activating site on rubisco (not active site!) ≡ CARBAMYLATION 3.pH increase favours carbamylation (H + sink in lumen) Rubisco is now catalytically ready to fix atmospheric CO2!

9. Plant cells also respire: convert O 2 →CO 2 1.Via mitochondiral respiration at night –This is oxidative phosphorylation to generate ATP in the dark (R on diagram) –This also happens in the light! 2.Via rubisco → can use O 2 as a substrate in photorespiration (PR) Therefore, measuring NET gas exchange in photosynthetic organs is difficult! We can define an apparent photosynthesis rate = CO 2 fixation rate – CO 2 evolution rate =gross p’syn – (mt R + PR) At a low atmospheric [CO 2 ] these values (GP) and (R+PR) are equal this is the CO 2 compensation point Fig. 5.16 (mt) (Rubisco O 2 -ase) (Rubisco CO 2 -ase) (mt)

10. Photorespiration is due to rubisco’s oxygenase activity Makes 2-phospho- glycolate (2C) + 3-PGA from RuBP + O 2 This C in 2-P- glycolate not wasted but reassimilated by exchange of intermediates with 2 other organelles –Peroxisome –Mitochondria Fig. 5.18 Exported or recycled to regenerate RuBP

11. The function of photorespiration is not immediately obvious Energetically wasteful, so why do it? Thoughts and theories… 1.[O 2 ] in atmosphere has been low during most of evolutionary history Therefore PR is an evolutionary relic? No! PR mutants are lethal! → Therefore, PR is essential No evolutionary pressure to get rid of O 2 -ase function 2.The salvage cycle does a good job of recovering photorespired C Each 2 turns of the 2-P-glycolate salvage cycle forms 4 3-PGA 1 lost, 3 returned to the PCR cycle Complex salvage pathway works well! 3.Metabolic safety valve? PR protects against photoxidative damage by allowing P.E.T. to continue at low [CO 2 ] e.g., under high light + low CO 2 (photoinhibitory conditions, stomata closed, water-stressed)

12. The chloroplast oxidative pentose phosphate cycle allows plants to make NADPH in the dark Shares intermediates with PCR Both at once: FUTILE CYCLE! –Use 3 ATP –No CO 2 fixation! Both pathways are light- regulated PCR a/k/a RPPC OPPC Light induces changes the structure of the disulfide bonds of the pathways’ enzymes –PCR cycle enzymes active when reduced –OPPC cycle enzymes active when oxidized LightDark PCR enzymes √X OPPC enzymes X√ Fig. 5.20 Why have an OPPC? –Make NADPH in dark –Make ribose and deoxyribose for nucleic acid synthesis

13. 1.Mesophyll fewer chloroplasts 2.Bundle sheath cells lots of chloroplasts surround vascular tissue thick cell walls prevent diffusion of CO 2 out of BS cells and traps photorespired CO 2 No mesophyll cells are more than 2-3 cells away from BS This ensures quick export of fixed CO 2 as sucrose Many chloroplasts needed to fix high [CO 2 ] How can plants minimize PR and maximize GP? Plants are separated into 2 main groups based on their ability to do this: Plants where 3-C 3-PGA is product of CO 2 fix’n = C3 Plants where 4-C oxaloacetate is product of CO 2 fix’n = C4 C4 plants have 2 distinct photosynthetic tissues –Leaf anatomy differs from C3 leaf Fig. 5.21: C4 leaf X-section (e.g., maize)

14. C4 plants are present in all 18 plant families This includes flowering plants as well C4 plants are: Better at CO 2 fixation (up to 3X more efficient) Better at drought stress Concentrate CO 2 at rubisco active site and thus minimize CO 2 loss! How do they do this? –Fix CO2 into C4 organic acid in the mesophyll cell using PEP carboxylase (not rubisco!) –Use a transporter to move the acid into the bundle sheath cell –Release CO 2 there –Fix CO 2 via PCR –Recycle C3 acid released (pyruvate) back to mesophyll PEP carboxylase malate pyruvate Malic Enzyme Fig. 5.22: the C4 carbon fixation pathway

15. C4 metabolism pluses: –CO 2 outcompetes O 2 at rubisco active site: Less PR! –Much lower compensation point maintain high CO 2 fixation rates when stomata are partially closed → conserves H 2 O –Lower transpiration ratio = less H 2 O transported per CO 2 assimilated Using the C4 pathway to fix carbon is not always an advantage for the plant C4 metabolism minuses: –Need to “spend” 2 ATP per CO 2 to recycle C3 acid back to the mesophyll cells C3 plants often have an ecological advantage –Grow better in cooler climates and low irradiance –Higher CO 2 assimilation rate in environments with lots of water

16. How do plants grow in the desert? Use CAM metabolism: conserves H 2 O CAM plants have an inverted stomatal cycle –Night: open –Day: closed Therefore CO 2 uptake at night → accumulate malate in vacuole During day → convert malate to starch via PCR cycle Need PEPC as in C4 photosynthesis –Requires lots of PEP (PEPC substrate), provided from glycolytic breakdown of starch CAM is similar to C4, but: –No specialized anatomy (specialized cell types) –No closed cycle of carbon intermediates Night Day PEPC Large, watery Decarbox- ylation Malic enzyme export Fig. 5.26

17. CAM plants are evolutionarily adapted to live in low water environments CAM plants have even lower transpiration ratios than C4 plants BUT –Only fix <1/2 C of C3 and <1/3 of C4 plants → slow growers –But can continue CO2 uptake under H2O stress Reassimilate respired CO2 Some plants can “switch on” CAM metabolism (facultative vs. obligatory) From http://www.arizonensis.org/images/plantae /cereus_gigant.jpg