Management of Reserves Alison 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 cellular components synthesis of reserves and their mobilisation regulation is essential

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

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?!

Synthesis of Starch and Sucrose Photosynthetic cell 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 PGA CO2 RuBP 1,3 bisPGA Triose P starch sucrose RuBP = ribulose 1,5-bis-phosphate (pentose) 3-PGA = 3-phosphoglycerate 1,3 bisPGA = 1,3 bis-phosphoglycerate

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

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

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

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

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

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

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

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?

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

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 Spinacia oleracea leaf 25.0 0.4 Lolium temulentum leaf 9.6 0.4 Pisum sativum root stele 4.6 26.4 Pisum sativum root cortex 1.6 5.2 14CO2 incorporation experiments show label goes from UDP-Glucose  sucrose-P  sucrose

Location of sucrose synthesis Subcellular fractionation of pea leaves Homogenize tissue in isotonic buffer Spin at 2000g 2000g pellet 2000g supernatant 36000g supernatant Spin at 36000g 36000g pellet 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

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 25 50 75 100 125 10 20 30 40 time (sec) hexose P 3 PGA nmol/mg chl Triose phosphate and 3-Pglycerate can cross envelope at rates comparable to photosynthesis

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

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) UTP PPi CH2OH OH O-P-O-P-O-uridine O O- Glucose 1-P UDP-G pyrophosphorylase

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

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

Starch Synthesis in vivo Hard to measure starch synthase and phosphorylase in vivo Phosphorylases act in degradative direction Arabidopsis starchless mutant 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 Glucose units added to non-reducing end, from ADP-G, forming a-1,4 links ADP-G starch synthase ADP

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

Mendel’s Wrinkled Peas wrinkled (rr) peas have less amylopectin than wt - collapse on drying 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 ‘Branching Enzyme’ Activity in embryos One isozyme of BE has 0.8 kbp transposon in r locus (rugosus) [Alison Smith, John Innes Centre]

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

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

Lecture 9 - Summary Sugars translocated Storage carbohydrate 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!

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