Page 1
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
Page 2
Management of Reserves Management of Reserves LecturesLectures
9. Carbohydrate synthesis, transport and storage
10. Lipid synthesis, mobilisation of reserves
11. Regulation of metabolism
Page 3
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?!
Page 4
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
Page 5
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
Page 6
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
Page 7
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) ++ + ++++ -
Page 8
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
Page 9
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
Page 10
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
Page 11
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
Page 12
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?
Page 13
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
Page 14
• 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
Page 15
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
Page 16
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
Page 17
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
Page 18
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
Page 19
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
Page 20
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
Page 21
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
Page 22
The First Plant BiochemistThe First Plant Biochemist
Gregor Mendel1822-1888 round versus wrinkled peas
Page 23
• 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
Page 24
Branching EnzymeBranching Enzyme
• Branching enzyme forms the -1,6 links
start
start
branching enzyme
starch synthase
-1,4 link
-1,6 link
Page 25
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
Page 26
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!
Page 27
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