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Carbohydrate Biosynthesis in Plants

Carbohydrate Biosynthesis in Plants. CH353 January 15, 2008. Overview of Plant Metabolism. Overview of Carbon Assimilation. Occurs in Chloroplasts Stage 1: Fixation 1 step – RUBISCO unique to plants Stage 2: Reduction 3 steps – analogous to gluconeogenesis (uses NADPH)

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Carbohydrate Biosynthesis in Plants

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  1. Carbohydrate Biosynthesis in Plants CH353 January 15, 2008

  2. Overview of Plant Metabolism

  3. Overview of Carbon Assimilation Occurs in Chloroplasts Stage 1: Fixation • 1 step – RUBISCO unique to plants Stage 2: Reduction • 3 steps – analogous to gluconeogenesis (uses NADPH) Stage 3: Regeneration • 9 steps; 7 enzymes analogous to pentose phosphate pathway

  4. Stages of Carbon Assimilation • Stage 1: Fixation Rubisco ribulose 1,5-bisphosphate + CO2→ 2 3-phosphoglycerate • Stage 2: Reduction 3-phosphoglycerate kinase 3-phosphoglycerate + ATP→ 1,3-bisphosphoglycerate + ADP glyceraldehyde 3-phosphate dehydrogenase 1,3-bisphosphoglycerate + NADPH → glyceraldehyde 3-phosphate + NADP+ + Pi triose phosphate isomerase glyceraldehyde 3-phosphate ↔ dihydroxyacetone phosphate

  5. Carbon Assimilation Stage 3:Regeneration of Acceptor

  6. Transketolase Reactions Donor1 Acceptor1 Acceptor2 Donor2 TPP • Transketolases transfer “active aldehyde” from a ketose (donor) to an aldose (acceptor) with cofactor thiamine pyrophosphate (TPP) • Transketolase reactions for carbon assimilation in chloroplast are identical to those for pentose phosphate pathway in cytosol or Sedoheptulose 7-phosphate or Ribose 5-phosphate

  7. Transaldolase Reaction Sedoheptulose 1,7-bisphosphate or • Transaldolases transfer dihydroxy-acetone phosphate (donor) to an aldose (acceptor) forming an aldol condensation adduct • Involves Schiff base enzyme bound intermediate • Transaldolase reaction (pictured) is identical to aldolase reaction in glycolysis/gluconeogenesis; other is unique to carbon assimilation • Donor: dihydroxyacetone phosphate • Acceptors: erythrose 4-phosphate and glyceraldehyde 3-phosphate ↓↑ + Donor Acceptor or Erythrose 4-phosphate

  8. Stage 3: Regeneration of Acceptor glyceraldehyde 3-phosphate transaldolase ↑↓ + dihydroxyacetone phosphate fructose 1,6-bisphosphate bisphosphatase ↓ - Pi fructose 6-phosphate transketolase ↑↓ + glyceraldehyde 3-phosphate erythrose 4-phosphate + xylulose 5-phosphate transaldolase ↑↓ + dihydroxyacetone phosphate sedoheptulose 1,7-bisphosphate bisphosphatase ↓ - Pi sedoheptulose 7-phosphate transketolase ↑↓ + glyceraldehyde 3-phosphate ribose 5-phosphate + xylulose 5-phosphate transaldolase has same ketose as substrate transketolase has same aldose as substrate bisphosphatases make process irreversible

  9. Stage 3: Regeneration of Acceptor 2 xylulose 5-phosphate 1 ribose 5-phosphate ribulose 5-phosphate epimerase ↑↓ ↑↓ ribose 5-phosphate isomerase 3 ribulose 5-phosphate ribulose 5-phosphate kinase ↓ + 3 ATP → 3 ADP 3 ribulose 1,5-bisphosphate Stage 3 Net: Input: 15 C Output: 15 C 2 dihydroxyacetone phosphate 3 ribulose 1,5-bisphosphate 3 glyceraldehyde 3-phosphate 3 ADP 3 ATP 2 Pi

  10. Stoichiometry of Carbon Assimilation Overall Process: 3 CO2 + 9 ATP + 6 NADPH → glyceraldehyde 3-phosphate + 9 ADP + 6 NADP+ + 8 Pi • Assimilation of 3 carbons and 1 phosphorous per cycle • Inorganic phosphate must be replaced for sustained ATP synthesis in chloroplast

  11. Phosphate–Triose Phosphate Antiporter • Exchanges dihydroxyacetone phosphate or 3-phosphoglycerate for phosphate • In light: triose phosphate transported to cytosol with antiport of phosphate to chloroplast stroma • Phosphate is released in cytosol with sucrose biosynthesis

  12. ATP and Reducing Equivalents Exchange • Exchange of ATP and reducing equivalents mediated by antiporter • only 3-phosphoglycerate or dihydroxyacetone phosphate transported • ATP and NADPH used on stromal side and ATP and NADH generated on cytosolic side • no net flux of phosphate or triose phosphate

  13. Regulation of Enzymes • Rubisco • Rubisco activase removes substrate from inactive enzyme (ATP hydrolyzed) • Carbamoylation of active site lysine (CO2 + Mg+2) • Nocturnal inhibitor binds • Photosynthetic environment in chloroplast stroma ↑ NADPH ↑ pH ↑ Mg2+ • Conditions stimulate enzyme activity • Rubisco activation (carbamoyllysine formation) is faster • Fructose 1,6-bisphosphatase activity ↑ 100x with illumination • Reduction of enzymes RS–SR’ → RSH + HSR’

  14. Regulation of Enzymes • Photosynthetic environment in chloroplast stroma ↑ NADPH ↑ pH ↑ Mg2+ Effect of pH and [Mg2+] on activity of fructose 1,6-bisphosphatase

  15. Regulation of Enzymes sulfhydryls (reduced) disulfides (oxidized) Activated by Reduction of Disulfides • glyceraldehyde 3-phosphate dehydrogenase • fructose 1,6-bisphosphatase • sedoheptulose 1,7-bisphosphatase • ribulose 5-phosphate kinase Inactivated by Reduction: • glucose 6-phosphate dehydrogenase

  16. Rubisco Oxygenase Activity • Rubisco accepts both CO2 and O2 as substrates • Incorporation of O2 into ribulose 1,5-bisphosphate produces: • 3-phosphoglycerate • 2-phosphoglycolate • No fixation of CO2 • Requires 2-phosphoglycolate salvage

  17. Glycolate Pathway • Salvage of 2-phosphoglycolate • Involves metabolite transport and enzymes in chloroplast, peroxisome and mitochondrion • Glycine decarboxylase is key enzyme • Process consumes O2 and evolves CO2 “Photorespiration” • Wastes energy and fixed carbon and nitrogen

  18. C4 Pathway • Rubisco oxygenase activity favored by high temperature/low moisture environments • C4 plants separate fixation of HCO3- and CO2 in different but metabolically-linked cells • Requires more energy (2 ATP’s) but avoids wasteful oxygenase reaction • CAM plants temporally separate 2 fixations (store malate at night)

  19. Starch Biosynthesis Carbohydrate storage Occurs in plastids ADP-glucose substrate Adds to reducing end (unlike glycogen synthesis) α(1→4) glucose (amylose) with α(1→6) branches (amylopectin) Sucrose Biosynthesis Carbohydrate transport Occurs in cytoplasm Fructose 6-phosphate & UDP-glucose Joins reducing (anomeric) hydroxyls Glucose(α1↔β2)Fructose Starch and Sucrose Biosynthesis • Excessive amounts of triose and monosaccharide phosphates are converted to alternative forms in the light • Liberates phosphate for ATP synthesis

  20. Cellulose Biosynthesis • Cell wall structure • Occurs in cytoplasm and at plasma membrane • Lipid-linked carrier and membrane protein complex • UDP-glucose is generated from sucrose and UDP by sucrose synthase • UDP-glucose is substrate for cellulose synthase; adds glucose monomers to non-reducing end • Cellulose isβ(1→4)linked glucose

  21. Regulation of Sucrose Biosynthesis • Need phosphate for ATP synthesis and triose phosphate for carbon fixation • Fructose 2,6-bisphosphate (F2,6BP) activates pyrophosphate-dependent phosphofructokinase-1 (PP-PFK-1) and inhibits fructose bisphosphatase-1 (FBPase-1) • Its synthesis by phosphofructokinase-2 is inhibited by triose phosphates (light) and activated by phosphate (dark) • In dark: ↑ Pi, ↑ F2,6BP, ↑ F1,6BP → glycolysis • In light: ↑ triose phosphates, ↓ F2,6BP, ↑ F6P → sucrose biosynthesis

  22. Regulation of Sucrose Biosynthesis • Sucrose 6-phosphate synthase (SPS) is partially inactivated by phosphorylation by SPS kinase • In light: glucose 6-phosphate (high gluconeogenesis) directly stimulates SPS and inhibits SPS kinase activating SPS (sucrose biosynthesis) • In dark: phosphate directly inhibits SPS and inhibits SPS phosphatase inactivating SPS (no sucrose biosynthesis)

  23. Regulation of Starch Biosynthesis ADP-glucose pyrophosphorylase synthesizes starch precursor • inhibited by high [Pi] accumulating in the dark (ATP hydrolysis) • activated by high [3-phosphoglycerate] accumulating in the light (carbon assimilation; diminished sucrose biosynthesis)

  24. Gluconeogenesis from Fats • Germinating seeds convert stored fats into sucrose • β-oxidation (glyoxysome) fatty acid → acetyl-CoA • glyoxylate cycle converts 2 acetyl-CoA → succinate • mitochondrial citric acid cycle & cytoplasmic gluconeogenesis converts succinate → hexoses

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