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Management of ReservesAlison Smith ([email protected])
CO2fixation produces triose P
conversion to sucrose for translocation to sink tissues
nutrient assimilation - N, S, Pand synthesis of allcellularcomponents
synthesis of reserves and their mobilisation
regulation is essential
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Management of ReservesLectures
9. Carbohydrate synthesis, transport and storage
10. Lipid synthesis, mobilisation of reserves
11. Regulation of metabolism
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chloroplast
Photosyntheticcell
Synthesis of Starch and Sucrose
sucrose
starch
1,3 bisPGA
PGACO2
RuBP
Triose P
Sucrose is principalphotosynthetic product
accounts for most of CO2absorbed
important storage sugar tap root of carrots and sugar
beet (up to 20% dry weight)
and in leaves, eg 25% leaf dryweight in ivy
major form fortranslocation of carbon
from photosynthetic leaves(source leaves)
in germinating seedlings afterstarch or lipid breakdown
RuBP = ribulose 1,5-bis-phosphate (pentose)
3-PGA = 3-phosphoglycerate
1,3 bisPGA = 1,3 bis-phosphoglycerate
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Sugar Translocation is Essential
Sugars required formetabolism
all the time, in all tissues
Sugars produced only
by source tissues in light period
Translocation occurs
source to sink over shortterm
from storage tissues toyoung tissues over longterm
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Sugar 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 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-photosynthetic cells
amyloplas t
Non-photosynthetic cell
long-term starch storage roots, tubers, seeds
starch
chloroplas t sucrose
Triose Pstarch
sucrose
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Importance of Starch
Sunflower after 47 min photosynthesis
Carbon absorbed (mg) 7.87Hexose accumulated 1.17
Sucrose 4.20
Starch 1.84
Starch is the dominant storage polysaccharide in most plants
In leaves - t ransitory starch- in chloroplasts
high percentage of CO2assimilated goes directly into starch
In nonphotosynthetic cells - storage starchin 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 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 m in diameter
rice: angular,
10 m in diameter
acceptors
for addition
of furtherglucose units
start
(reducing end)
polymer of glucose units
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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
Bu t store starch in the seed
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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 Metabolism
Sucrose
UDP-Glucose Fructose 6P
Sucrose P
Pi
Sucros e P Synth ase
Sucros e P
Phosphatase
UDP
Fructose
+UDP-Glucose
Sucros e SynthaseUDP
Fructose
+Glucose
Invertase
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Relationship between enzyme activities and sucrose synthesis
Sucrose is made via Sucrose-P
14CO2incorporation experiments show label goes from
UDP-Glucose sucrose-P sucrose
Activity (mol/hr per g FW)
Tissue Sucrose P Sucrosesynthase synthase
Spinaciaoleracea leaf 25.0 0.4
Lol iumtemulentum leaf 9.6 0.4
Pisumsat ivum root stele 4.6 26.4
Pisumsat ivum root cortex 1.6 5.2
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Location of sucrose synthesis
Subcellular fractionation of pea leaves
Homogenize tissue in isotonic buffer
36000g
supernatant
Spin at 36000g
36000g pellet
Spin at 2000g
2000gpellet 2000gsupernatant
Activity per fraction (mol/h)
Cell fraction SPS Rubisco
2000gpellet 0.74 273
36000gpellet 2.6 74
Supernatant 19.3 350
ie SPS is in the cytosol
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Permeability 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
nmol/mgch
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
Competition experiments
uptake of 3-carbon compounds by same carrier strict counter exchange for Pi
Export of carbon
Major compound exported is DHAP20X more than Ga3P
3-PGA taken up
Pi released
0
50
100
150
0 100 200 300 400
time (sec)
nmol/mg
chl
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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)
CH2OH
OH O-P-O-P-O-uridine
OH
OH
O
O- O-
OOGlucose 1-P
UDP-G
pyrophosphory lase
UTP
PPi
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Pathway of sucrose synthesis from CO2
DHAPCO2
Ga3P
3PGA
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 Synthesis
1,4 glucann+1Glucose 1-P
1,4 glucann
Starch
phosphorylase
Pi
ADPglucose
Starch
synthase
1,4 glucann
Starch
Glucose 1-P
PPi
ATP
ADPG PPiase
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Starch Synthesis in v ivo
Hard to measure starch synthase and phosphorylase
in v ivo 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 Biochemist
Gregor Mendel
1822-1888 round versus wrinkled peas
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wrinkled(rr) peas have less amylopectin than wt - collapseon drying
Mendels Wrinkled Peas
One isozyme of BE has 0.8 kbp transposon in rlocus
(rugosus)[Al iso n Smith , Joh n Innes Centre]
Weight (mg) BE (mol.min-1) Starch (mg)
RR rr RR rr
100 0.89 0 4 0.8
200 2.98 0 20 2.2300 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 Enzyme
Branching enzyme forms the -1,6 links
start
start
branching enzyme
starch synthase
-1,4 link
-1,6 link
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chloroplas t
source cell
Synthesis of Transitory Starch
sucrose
starch
ADP-G PPiase, Starch synthaseand BE all found in chloroplast
export tophloem
PGA
1,3 bisPGA
Triose P
CO2
RuBP
L t 9 S
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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!
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Genetic or biochemical modifications ofstarch are or may be used for...
Modified starch
Phosphate content
water absorbency
improve starchgranule integrity
(cross linker)
modified
starchfried 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