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

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 whats happening?!

chloroplast
Photosynthetic cell
Synthesis of Starch and Sucrose
sucrose
starch
1,3 bisPGA
PGA
CO
2
RuBP
Triose P
Sucrose is principal
photosynthetic product
accounts for most of CO
2

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-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


CH
2
OH CH
2
OH
| |
HCOH HOCH
| |
HOCH HOCH
| |
HCOH HCOH
| |
HCOH HCOH
| |
CH
2
OH CH
2
OH

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
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 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) ++ + ++++ -
Photosynthetic cell
transitory starch storage
green leaves
Starch is made in photosynthetic and non-
photosynthetic cells
amyloplast
Non-photosynthetic cell
long-term starch storage
roots, tubers, seeds
starch
chloroplast
sucrose
Triose P
starch
sucrose
Importance of Starch
Sunflower after 47 min photosynthesis

Carbon absorbed (mg) 7.87
Hexose accumulated 1.17
Sucrose 4.20
Starch 1.84
Starch is the dominant storage polysaccharide in most plants

In leaves - transitory starch - in chloroplasts
high percentage of CO
2
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

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 further
glucose units
start
(reducing end)
polymer of glucose units
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
Sucrose
UDP-Glucose Fructose 6P
Sucrose P
Pi
Sucrose P Synthase
Sucrose P
Phosphatase
UDP
Fructose
+
UDP-Glucose
Sucrose Synthase
UDP
Fructose
+
Glucose
Invertase
Relationship between enzyme activities and sucrose synthesis
Sucrose is made via Sucrose-P

14
CO
2
incorporation experiments show label goes from
UDP-Glucose sucrose-P sucrose
Activity (mol/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
Location 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 (mol/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

0
25
50
75
100
125
0 10 20 30 40
time (sec)
hexose P
3 PGA
n
m
o
l
/
m
g

c
h
l

Triose phosphate and 3-Pglycerate can
cross envelope at rates comparable to
photosynthesis

CH
2
OP CH
2
OP CH
2
OP
| | |
CHOH C=O CHOH
| | |
COOH CH
2
OH CHO

3-P glycerate DHAP Glyceraldehyde
3-P
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
0 100 200 300 400
time (sec)
n
m
o
l
/
m
g

c
h
l

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)
CH
2
OH
OH
O-P-O-P-O-uridine
OH
OH
O
O
-
O
-

O O
Glucose 1-P
UDP-G
pyrophosphorylase
UTP
PPi
Pathway of sucrose synthesis from CO
2
DHAP
CO
2
Ga3P
3PGA
RuBP
1,3 bisPGA
CO
2
FBP
Ga3P
sucrose P
F6P
G6P
Pi
DHAP
sucrose
UTP
PPi
G1P
UDGP
Enzymes of Starch Synthesis
1,4 glucan
n+1

Glucose 1-P
1,4 glucan
n

Starch
phosphorylase
Pi
ADPglucose
Starch
synthase
1,4 glucan
n

Starch
Glucose 1-P
PPi
ATP
ADPG PPiase
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
F
1
(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-G
starch
synthase
The First Plant Biochemist
Gregor Mendel
1822-1888
round versus wrinkled peas
wrinkled (rr) peas have less amylopectin than wt - collapse
on drying
Mendels Wrinkled Peas
One isozyme of BE has 0.8 kbp transposon in r locus
(rugosus)
[Alison Smith, J ohn 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 2.2
300 3.95 0.56 60 5.4
400 7.81 1.12 86 16.5

Branching Enzyme Activity in embryos
Branching Enzyme
Branching enzyme forms the -1,6 links

start
start
branching enzyme
starch synthase
-1,4 link
-1,6 link
chloroplast
source cell
Synthesis 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
CO
2
RuBP
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!

Genetic or biochemical modifications of
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