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) Stage 3: Regeneration 9 steps; 7 enzymes analogous to pentose phosphate pathway

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

Carbon Assimilation Stage 3: Regeneration of Acceptor

Transketolase Reactions Donor1 Acceptor1 Acceptor2 Donor2 TPP or Sedoheptulose 7-phosphate or Ribose 5-phosphate 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

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

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 sedoheptulose 7-phosphate ribose 5-phosphate + xylulose 5-phosphate transaldolase has same ketose as substrate transketolase has same aldose as substrate bisphosphatases make process irreversible

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

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

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

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

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’

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

Regulation of Enzymes Activated by Reduction of Disulfides 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

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

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

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)

Starch and Sucrose Biosynthesis Excessive amounts of triose and monosaccharide phosphates are converted to alternative forms in the light Liberates phosphate for ATP synthesis 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

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 is β(1→4) linked glucose

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

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)

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)

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