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Management of ReservesManagement of Reserves Alison Smith ([email protected])Alison Smith ([email protected])
• 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
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Management of Reserves Management of Reserves LecturesLectures
9. Carbohydrate synthesis, transport and storage
10. Lipid synthesis, mobilisation of reserves
11. Regulation of metabolism
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Lecture 9 - Lecture 9 - Carbohydrate Synthesis, Transport and Carbohydrate Synthesis, Transport and
StorageStorage
• Sugars produced by plant
• Pathway of sugar synthesis from photosynthate
• How do you work out what’s happening?!
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chloroplast
Photosynthetic cell
Synthesis of Starch and SucroseSynthesis of Starch and Sucrose
sucrose
starch
1,3 bisPGA
PGACO2
RuBP
Triose P
• 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
RuBP = ribulose 1,5-bis-phosphate (pentose)3-PGA = 3-phosphoglycerate1,3 bisPGA = 1,3 bis-phosphoglycerate
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Sugar Translocation is EssentialSugar 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
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Sugar translocationSugar translocation
• Analysis of phloem sap– shallow incision produces little sap
CH2OH CH2OH| |
HCOH HOCH| |
HOCH HOCH| |
HCOH HCOH| |
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
- severed aphid stylets most effective!
stylet bundle cut by laser or radiofrequency microcautery
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Sugar composition of phloem sapSugar composition of phloem sap
• > 500 different species (100 families) of dicots (Zimmermann & Ziegler, 1975)
• most families transport sucrose
• concentration in phloem sap can reach 1 M
Sucrose Raffinose Stachyose Sugar alcoholsMost 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) ++ + ++++ -
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Photosynthetic cell• transitory starch storage• green leaves
Starch is made in photosynthetic and non-Starch is made in photosynthetic and non-photosynthetic cellsphotosynthetic cells
amyloplast
Non-photosynthetic cell• long-term starch storage• roots, tubers, seeds
starch
chloroplast sucrose
Triose Pstarch
sucrose
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Importance of StarchImportance of Starch
Sunflower after 47 min photosynthesis
Carbon absorbed (mg) 7.87Hexose accumulated 1.17Sucrose 4.20Starch 1.84
• Starch is the dominant storage polysaccharide in most plants
• In leaves - transitory starch - in chloroplasts– high percentage of CO2 assimilated goes directly into starch
• 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
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Composition of StarchComposition of Starch
• Amylose– -1,4-glucan
– ~1000 glucose units
• Starch grain – Water insoluble,
– size & shape is species specific
• Amylopectin– -1,4 & -1,6-glucan– 10,000 - 100,000 glucose units– highly branched, 20 - 25 glucoses/branch
potato: oval, 100 µm in diameter
rice: angular,10 µm in diameter
acceptors for additionof furtherglucose units
start(reducing end)
polymer of glucose units
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FructansFructans
• 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
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How are Sucrose and Starch Synthesised?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?
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Enzymes of Sucrose MetabolismEnzymes of Sucrose Metabolism
Sucrose
UDP-Glucose Fructose 6P
Sucrose P
Pi
Sucrose P Synthase
Sucrose PPhosphatase
UDP
Fructose +UDP-Glucose
Sucrose SynthaseUDP
Fructose +Glucose
Invertase
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• Relationship between enzyme activities and sucrose synthesis
Sucrose is made via Sucrose-PSucrose is made via Sucrose-P
• 14CO2 incorporation experiments show label goes from
UDP-Glucose sucrose-P sucrose
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
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Location of sucrose synthesisLocation of sucrose synthesis
• Subcellular fractionation of pea leaves
Homogenize tissue in isotonic buffer
36000g supernatant
Spin at 36000g
36000g pellet
Spin at 2000g
2000g pellet 2000g supernatant
Activity per fraction (µmol/h)
Cell fraction SPS Rubisco
2000g pellet 0.74 27336000g pellet 2.6 74Supernatant 19.3 350
ie SPS is in the cytosol
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Permeability of Chloroplast EnvelopePermeability of Chloroplast Envelope
• How does fixed carbon get to the cytosol?– Uptake of labelled compounds into isolated chloroplasts
0
25
50
75
100
125
0 10 20 30 40
time (sec)
hexose P
3 PGA
nm
ol/
mg
ch
l
Triose phosphate and 3-Pglycerate can cross envelope at rates comparable to photosynthesis
CH2OP CH2OP CH2OP | | | CHOH C=O CHOH | | | COOH CH2OH CHO
3-P glycerate DHAP Glyceraldehyde 3-P
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Phosphate Translocator 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
3-PGA taken up
Pi released
0
50
100
150
0 100 200 300 400time (sec)
nm
ol/
mg
ch
l
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Synthesis of UDP-GlucoseSynthesis of UDP-Glucose
• Triose P is converted to hexose P by gluconeogenesis
• This must be converted to substrate for sucrose P synthase (UDP-G)
CH2OH
OH O-P-O-P-O-uridineOH
OH
O
O- O-
OOGlucose 1-P
UDP-G pyrophosphorylase
UTP
PPi
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Pathway of sucrose synthesis from COPathway of sucrose synthesis from CO22
DHAPCO2
Ga3P3PGA
RuBP
1,3 bisPGA
CO2
FBP
Ga3P
sucrose P
F6P
G6P
Pi
DHAP
sucrose
UTP
PPi
G1P
UDGP
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Enzymes of Starch SynthesisEnzymes of Starch Synthesis
1,4 glucann+1Glucose 1-P
1,4 glucann
Starch phosphorylase
Pi
ADPglucose
Starch synthase
1,4 glucann
Starch
Glucose 1-PPPi
ATP
ADPG PPiase
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Starch Synthesis Starch Synthesis in vivoin 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 -1,4 links
ADP
ADP-Gstarch synthase
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The First Plant BiochemistThe First Plant Biochemist
Gregor Mendel1822-1888 round versus wrinkled peas
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• wrinkled (rr) peas have less amylopectin than wt - collapse on drying
Mendel’s Wrinkled PeasMendel’s Wrinkled Peas
• One isozyme of BE has 0.8 kbp transposon in r locus (rugosus) [Alison Smith, John Innes Centre]
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
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Branching EnzymeBranching Enzyme
• Branching enzyme forms the -1,6 links
start
start
branching enzyme
starch synthase
-1,4 link
-1,6 link
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chloroplast
source cell
Synthesis of Transitory StarchSynthesis of Transitory Starch
sucrose
starch
• ADP-G PPiase, Starch synthase and BE all found in chloroplast
export to phloem
PGA
1,3 bisPGA
Triose P
CO2
RuBP
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Lecture 9 - SummaryLecture 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!
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Genetic or biochemical modifications of Genetic or biochemical modifications of starch are or may be used for...starch are or may be used for...
Modified starch
• Phosphate content• water absorbency• improve starch
granule integrity
(cross linker)
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