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Management of Reserves Alison Smith (as25@cam.ac.uk)

Management of Reserves Alison Smith (as25@cam.ac.uk). CO 2 fixation produces triose P conversion to sucrose for translocation to sink tissues nutrient assimilation - N, S, P – and synthesis of all cellular components synthesis of reserves and their mobilisation regulation is essential.

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Management of Reserves Alison Smith (as25@cam.ac.uk)

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  1. Management of ReservesAlison Smith (as25@cam.ac.uk) • CO2 fixation produces triose P • conversion to sucrose for translocation to sink tissues • nutrient assimilation - N, S, P – and synthesis of all cellularcomponents • synthesis of reserves and their mobilisation • regulation is essential

  2. Management of Reserves Lectures 9. Carbohydrate synthesis, transport and storage 10. Lipid synthesis, mobilisation of reserves 11. Regulation of metabolism

  3. Lecture 9 - Carbohydrate Synthesis, Transport and Storage • Sugars produced by plant • Pathway of sugar synthesis from photosynthate • How do you work out what’s happening?!

  4. CO2 PGA RuBP 1,3 bisPGA starch Triose P sucrose Synthesis of Starch and Sucrose Photosyntheticcell • Sucrose is principal photosynthetic product • accounts for most of CO2 absorbed • important storage sugar • tap root of carrots and sugar beet (up to 20% dry weight) • and in leaves, eg 25% leaf dry weight in ivy • major form for translocation of carbon • from photosynthetic leaves (source leaves) • in germinating seedlings after starch or lipid breakdown chloroplast RuBP = ribulose 1,5-bis-phosphate (pentose) 3-PGA = 3-phosphoglycerate 1,3 bisPGA = 1,3 bis-phosphoglycerate

  5. Sugar Translocation is Essential • Sugars required for metabolism • all the time, in all tissues • Sugars produced only • by source tissues • in light period • Translocation occurs • source to sink over short term • from storage tissues to young tissues over long term

  6. - severed aphid stylets most effective! stylet bundle cut by laser or radiofrequency microcautery Sugar alcohols (Polyols) CH2OH CH2OH | | HCOH HOCH | | HOCH HOCH | | HCOH HCOH | | HCOH HCOH | | CH2OH CH2OH Sorbitol (Rosaceae) Mannitol (Combretaceae) Sugar translocation • Analysis of phloem sap • shallow incision produces little sap Sugars Sucrose glucose-fructose (G-F) Raffinose G-G-F Stachyose Galactose-G-G-F

  7. Sugar composition of phloem sap • > 500 different species (100 families) of dicots(Zimmermann & Ziegler, 1975) Sucrose Raffinose Stachyose Sugar alcohols Most families ++++ + + - Aceraceae (maple) ++++ Tr Tr - Anacardiaceae (cashew) +++ Tr Tr - Asteraceae (aster) + Tr Tr - Betulaceae (birch) ++++ ++ ++ - Buddleiaceae (butterfly bush) ++ +++ ++++ - Caprifoliaceae (honeysuckle) +++ ++ Tr - Combretaceae (white mangrove) +++ ++ + +++ Fabaceae (legume) ++++ Tr Tr - Fagaceae (beech & oak) ++++ Tr Tr - Moraceae (fig) ++++ + ++ - Oleaceae (olive) ++ ++ +++ - Rosaceae (rose) +++ Tr Tr ++++ Verbenaceae (verbena) ++ + ++++ - • most families transport sucrose • concentration in phloem sap can reach 1 M

  8. starch Triose P sucrose chloroplast sucrose • Non-photosynthetic cell • long-term starch storage • roots, tubers, seeds starch amyloplast Starch is made in photosynthetic and non-photosynthetic cells • Photosynthetic cell • transitory starch storage • green leaves

  9. Importance of Starch • Starch is the dominant storage polysaccharide in most plants • In leaves - transitory starch - in chloroplasts • high percentage of CO2 assimilated goes directly into starch Sunflower after 47 min photosynthesis Carbon absorbed (mg) 7.87 Hexose accumulated 1.17 Sucrose 4.20 Starch 1.84 • In nonphotosynthetic cells - storage starch in amyloplasts • storage organs bananas, tubers (up to 80% dry weight), cereal grains (75% dry weight) • herbaceous roots, underground stems, bulbs perennials • trees young twigs, roots, parenchyma of bark xylem & phloem

  10. acceptors for addition of further glucose units polymer of glucose units start (reducing end) potato: oval, 100 µm in diameter rice: angular, 10 µm in diameter Composition of Starch • Amylopectin •  -1,4 &  -1,6-glucan • 10,000 - 100,000 glucose units • highly branched, 20 - 25 glucoses/branch • Amylose •  -1,4-glucan • ~1000 glucose units • Starch grain • Water insoluble, • size & shape is species specific

  11. Leaves of temperate Gramineae • C3 grasses - barley, oats, rye grass • major feedstuff for cattle & sheep in temperate zones Fructans • Most common are the fructans • water-soluble, non reducing polymers of fructose • 5 - 300 fructose units, joined to one glucose • Some plants store other compounds • Leaves, flowers and underground storage organs • Asteraceae (dahlias, jerusalem artichokes) • Liliaceae (onions, asparagus) • Iridaceae (irises) • But store starch in the seed

  12. How are Sucrose and Starch Synthesised? • Elucidated by careful biochemical studies • Determination of enzyme activities • what reactions are catalysed, thermodynamic considerations • Correlation with flux through pathways • is the activity which can be measured sufficient to account for the process taking place?

  13. Fructose 6P UDP-Glucose Sucrose P Synthase UDP Sucrose P Sucrose P Phosphatase Pi Invertase Fructose + Glucose Sucrose Synthase UDP Fructose + UDP-Glucose Enzymes of Sucrose Metabolism Sucrose

  14. Sucrose is made via Sucrose-P • Relationship between enzyme activities and sucrose synthesis • Activity (µmol/hr per g FW) • Tissue Sucrose P Sucrose • synthase synthase • Spinaciaoleracea leaf 25.0 0.4 • Loliumtemulentum leaf 9.6 0.4 • Pisumsativum root stele 4.6 26.4 • Pisumsativum root cortex 1.6 5.2 • 14CO2 incorporation experiments show label goes from • UDP-Glucose  sucrose-P  sucrose

  15. Spin at 2000g 2000g pellet 2000g supernatant Spin at 36000g 36000g pellet 36000g supernatant Location of sucrose synthesis Homogenize tissue in isotonic buffer • Subcellular fractionation of pea leaves Activity per fraction (µmol/h) Cell fraction SPS Rubisco 2000g pellet 0.74 273 36000g pellet 2.6 74 Supernatant 19.3 350 ie SPS is in the cytosol

  16. 3 PGA hexose P nmol/mg chl 125 time (sec) 100 75 50 25 0 0 10 20 30 40 Permeability of Chloroplast Envelope • How does fixed carbon get to the cytosol? • Uptake of labelled compounds into isolated chloroplasts CH2OP CH2OP CH2OP | | | CHOH C=O CHOH | | | COOH CH2OH CHO 3-P glycerate DHAP Glyceraldehyde 3-P Triose phosphate and 3-Pglycerate can cross envelope at rates comparable to photosynthesis

  17. 150 100 nmol/mg chl 50 Pi released 3-PGA taken up 0 0 100 200 300 400 time (sec) Phosphate Translocator • Competition experiments • uptake of 3-carbon compounds by same carrier • strict counter exchange for Pi • Export of carbon • Major compound exported is DHAP – 20X more than Ga3P

  18. UTP PPi CH2OH O OH O-P-O-P-O-uridine OH OH O- O- O O Synthesis of UDP-Glucose • Triose P is converted to hexose P by gluconeogenesis • This must be converted to substrate for sucrose P synthase (UDP-G) Glucose 1-P UDP-G pyrophosphorylase

  19. Ga3P DHAP FBP Pi F6P G6P sucrose P sucrose G1P UTP PPi UDGP Pathway of sucrose synthesis from CO2 CO2 RuBP DHAP CO2 3PGA Ga3P 1,3 bisPGA

  20. ADPglucose  1,4 glucann ADPG PPiase Starch synthase ATP Glucose 1-P PPi  1,4 glucann Glucose 1-P Pi Starch phosphorylase Enzymes of Starch Synthesis  1,4 glucann+1 Starch

  21. starch synthase ADP-G ADP Starch Synthesis in vivo • Hard to measure starch synthase and phosphorylase in vivo • Phosphorylases act in degradative direction • Arabidopsis starchless mutant • Glucose units added to non-reducing end, from ADP-G, forming a-1,4 links Plants ADPG PPiase Starch Wild type 54 ± 5 7.3 ± 0.4 Mutant 0 0 F1 (WT x Mutant) 50 ± 4 6.0 ± 0.5

  22. round versus wrinkled peas The First Plant Biochemist Gregor Mendel 1822-1888

  23. ‘Branching Enzyme’ Activity in embryos Weight (mg) BE (µmol.min-1) Starch (mg) RR rr RR rr 100 0.89 0 4 0.8 200 2.98 0 20 2.2 300 3.95 0.56 60 5.4 400 7.81 1.12 86 16.5 Mendel’s Wrinkled Peas • wrinkled (rr) peas have less amylopectin than wt - collapse on drying • One isozyme of BE has 0.8 kbp transposon in r locus (rugosus) [Alison Smith, John Innes Centre]

  24. start starch synthase start a-1,4 link branching enzyme a-1,6 link Branching Enzyme • Branching enzyme forms the -1,6 links

  25. source cell CO2 PGA RuBP 1,3 bisPGA starch Triose P sucrose export to phloem chloroplast Synthesis of Transitory Starch • ADP-G PPiase, Starch synthase and BE all found in chloroplast

  26. Lecture 9 - Summary • Sugars translocated • Mainly sucrose, but also other tri- and tetrasaccharides and sugar alcohols • Storage carbohydrate • starch or fructans • Elucidating a metabolic pathway • determining enzyme activities, correlating with flux measurements • Subcellular location • cell fractionation • Regulation • there must be some!

  27. + amylose + amylopectin modified starch • Improve freeze- • thaw of frozen food • paper strength • adhesive • livestock feed • addition • fried snacks • (crispness / browning) • thickener / • gelling agent • biodegradable • packing material • film coating • Phosphate content • water absorbency • improve starch • granule integrity • (cross linker) Genetic or biochemical modifications of starch are or may be used for... Modified starch

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