Biology Department

1. Biochemical fixation of CO2 by plants and its efficiency Jörg Schwender Biology Department CHEMRAWN-XVII & ICCDU-IX July 2007

2. Outline • Biochemistry of carbon fixation and the world’s most abundant protein 2) CO2 recycling mechanism in oilseeds (Rapeseed, Canola)

3. Fossil Fuels from Ancient Biomass Ancient forests Ancient phytoplankton www.fieldmuseum.org eobglossary.gsfc.nasa.gov Organic deposits Coal Petroleum Gas Special geological conditions, heat, pressure 1 Yr global fossil fuel consumption equals >400 Yr ancient primary production ! Dukes, Burning buried sunshine: human consumption of ancient solar energy. Climatic Change 61: 31–44, 2003

4. Primary Production of the Biosphere Photosynthesis Atmospheric / aquatic CO2 Organic compounds Global annual primary production is estimated as: 5.5 1010 tCarbon (terrestrial) 4.5 1010 tCarbon (oceanic) One predominant biochemical mechanism ! Field et al., (1998) Science 281, 237-240

5. Biochemistry of Primary Production (Photosynthesis) “Light reaction” “Dark reaction” H2O CO2 Biochemical energy currency hv ATP Light absorption Photolysis of water Photosynthetic electron transport Carboxylation Reduction Substrate regeneration NADPH O2 Sugars, Organic compounds

6. RubisCO – The Enzyme That Binds CO2 (Ribulose-1,5-bisphosphate carboxylase / oxygenase) CO2 CH O P 2 H C OH CH O P CH O P CH O P 2 2 2 COOH C O HO C OH C COO- H C OH C OH C O COOH H C OH H C OH H C OH H C OH CH O P CH O P CH O P 2 2 2 CH O P 2 D-phosphoglyceric acid (PGA) Ribulose 1,5-bisphosphate carboxylation 2 x C3 product C5 substrate C1 substrate

7. Reduction of Chemically Fixed Carbon HC=O COOH H C OH H C OH CH O P CH O P NADPH 2 2 D-phosphoglyceric acid (PGA) D-glyceraldehyde 3-phosphate (GAP) ATP Carboxylation product Simple sugar Main energy investment during photosynthesis !

8. 5 x C3 C6 C5 C4 3 x C5 5 x C3 NADPH RubisCO C5 ATP C7 C5 C5 C5 sugars 3 x CO2 6 x C3 Calvin Cycle: Regeneration of the C5 Substrate 9 additional enzymes Ribulose 1,5-bisphosphate Melvin Calvin, Nobel Prize in Chemistry 1961

9. RubisCO – The Enzyme That Binds CO2 (Ribulose-1,5-bisphosphate carboxylase / oxygenase) • Globally binds ~1011 tons of CO2 per year • 16 subunit protein complex • Most abundant protein on earth ! • ~60 % of protein in a green leaf Mol. Size Marker Leaf extract L subunit S subunit RubisCO, 3D protein structure (L8S8)

10. RubisCO Paradox (Ribulose-1,5-bisphosphate carboxylase / oxygenase) • Very slow enzyme ! • (One RubisCO molecule can only fix a few CO2 per seconds) • Cannot distinguish very well between CO2 and O2 as substrates ! COOH COOH CO2 carboxylase H C OH H C OH CH O P CH O P CH O P 2 2 2 C O PGA PGA H C OH H C OH COOH CH O P oxygenase 2 COOH O2 H C OH CH O P CH O P 2 2 PGA P-glycolate Millard et al., New Phytologist (2007) 175: 11–28

11. Biochemistry of RubisCO: CO2 or O2 as Substrates (0.038 %) CO2 Calvin-Benson Cycle Sugars, Complex organic compounds 3 ATP 2 NADPH CH O P 2 C O H C OH Photorespiratory pathway + re-assimilation of P-glycolate H C OH CH O P 2 2 NADPH 3.33 ATP O2 COOH (21 %) COOH H C OH CH O P CH O P 2 2 PGA P-glycolate As high as 50 % of energy is lost in Photorespiratory pathway ! Bowes et al., Biochem Biophys Res Comm 45,716-722 (1971)

12. Fix for Ineffective Photorespiration? Bacterial glycolate catabolic pathway added Improved photosynthesis, Increased biomass production Engineered “Photorespiratory Bypass” in Arabidopsis thaliana Kebeish et al., Nature Biotech. 25: 593-599 (2007)

13. Nature’s Fix: CO2 Concentration Mechanisms (CCM) “Turbocharger” for RubisCO CO2 C4 acid RubisCO Calvin Cycle ATP ATP NADPH Ambient CO2 No photorespiration ! (0.038 %) C4 Photosynthesis: High-light & temperature adaptation (Maize, Sugarcane …) About 30 % of global photosynthesis is C4 type

14. How Do Plants React to Elevated Atmospheric CO2 ? • At 380 ppm C3 Photosynthesis is not CO2 saturated • Free Air Carbon Enrichment (FACE) experiments: • Simulate increased CO2. • Short term experiments: increased photosynthesis observed ! • However, this increase is restricted and modulated in a complex • way by different environmental variables !

15. Part 2: Biochemical CO2 recycling For plants atmospheric CO2 is precious !

16. Fossil reserves Products Energy Refinery or Power Plant Today: Utilizing Fossil Reserves Ancient Plants • Greenhouse Gasses/Global Warming • Cost of fossil reserves increasing, cost of plant oils decreasing

17. Sunlight CO2 Product Tomorrow: Can We Make Reduced Carbon Feedstocks in “Green Plants”? Enzymes can convert reduced carbon into any desired form • Need to find or make enzymes to do these jobs

18. Rapeseed, Canola (Brassica napus L.) • Oil crop, world wide 3rd in production. • Edible oils • “Biodiesel”, renewable alternative to fossil fuels • Source of special fatty acids for Industrial use • Biochemistry of developing seeds very well studied

19. Developing Seeds inside Siliques Developing embryo accumulates oil • Uptake of sucrose, • amino acids • 20 % of ambient • light reaches the • seeds • Photo-heterotrophic • growth JBC Nov. 10, 2006 Schwender et al., JBC 281(45): 34040-34047 (2006)

20. Assumed Carbon Economy of Oil Storing Seeds Oil storing seed hv Sucrose (glycolysis) Photosynthesis COOH Pyruvate C O CH 3 CO2 CO2 1/3 of Carbon lost as CO2 ! CoAS C O Acetyl-CoA CH 3 Fatty acids

21. Activation of RubisCO under in Planta Light Conditions RuBisCO: Protein abundant & activated Sufficient activity to support oil synthesis Substrate (CO2) abundant Ruuska et al. Plant Physiol. 136: 2700-2709 (2004) RubisCO is highly active in B. napus developing seeds !

22. Embryosin Culture: Observe Metabolism under Fixed Conditions Light CO2 Sucrose Glucose Central Metabolism Alanine Protein Glutamine Oil 1 mm What’s the path of carbon to Oil synthesis ?

23. 13CO2 CH O P 2 H C OH CH O P 2 13COOH C O H C OH COOH H C OH H C OH CH O P 2 CH O P 2 D-phosphoglyceric acid (PGA) Ribulose 1,5-bisphosphate Key Experimental Observations for Embryos • Unexpected low CO2 emission rate (less than 1/3 of C lost) • Incorporation of 13CO2, (RubisCO) but no randomization of positional label according to Calvin Cycle !

24. Flux Modes Analysis of Enzyme System Mode C explains experimental observations ! Schwender et al. (2004) Nature 432, 779–782

25. 6 C5 Enzymes of Calvin Cycle 6 CO2 RubisCO Glycolysis 12 Pyruvate 10 Pyruvate 12 Ac-CoA 6 CO2 10 Ac-CoA 10 CO2 6C 24C Storage Oil Carbon Balance of Glycolysis and RubisCO Bypass Sucrose 30C 5 C6 Glycolysis RuBisCo in non-cyclic context 10 Pyruvate 10 Ac-CoA 10 CO2 20C 10C Storage Oil Lost !

26. CO2 recycling in the Fruit Structure (Pod) Embryo Silique wall Sucrose CO2 Seed CO2 Seed coat 1 mm Nature 432, 684 (2004) 20 -23 Days After Flowering

27. Summary / Outlook 2) • Developing oilseed: • RubisCO takes part in sugar – to – oil conversion in a unexpected metabolic context. • Better carbon economy of seed storage synthesis • Key to understand & influence carbon partitioning into different storage compounds in Rapeseed and similar seeds. • Key to Yield Current Research: Analyze central metabolism in oil producing seeds and microorganisms • RubisCO fixes atmospheric CO2, is part of a catalytic cycle (Calvin cycle) 1) • RubisCO is highly abundant, somehow ineffective/inaccurate • CO2 Concentration Mechanisms (CCM’s)