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
chloroplast – accounts for most of CO2
CO2
PGA absorbed

RuBP
1,3 bisPGA • important storage sugar
– tap root of carrots and sugar
beet (up to 20% dry weight)
– and in leaves, eg 25% leaf dry
starch Triose P weight in ivy

• major form for

sucrose 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 - severed aphid stylets most effective!
– shallow incision produces little sap

stylet bundle cut by laser or radiofrequency microcautery

Sugar alcohols (Polyols)

CH2OH CH2OH
| |
HCOH HOCH
| | Sugars
HOCH HOCH Sucrose glucose-fructose (G-F)
| |
HCOH HCOH Raffinose G-G-F
| | Stachyose Galactose-G-G-F
HCOH HCOH
| |
CH2OH CH2OH

Sorbitol (Rosaceae) Mannitol (Combretaceae)

 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

Photosynthetic cell
• transitory starch storage
• green leaves
starch Triose P

chloroplast sucrose

Non-photosynthetic cell sucrose

• long-term starch storage
• roots, tubers, seeds
starch
amyloplast
 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
–  -1,4 &  -1,6-glucan –  -1,4-glucan
– 10,000 - 100,000 glucose units – ~1000 glucose units
– highly branched, 20 - 25 glucoses/branch

acceptors
polymer of glucose units for addition
of further
start
glucose units
(reducing end)

• Starch grain
– Water insoluble,
– size & shape is
species specific

potato: oval, rice: angular,

100 µm in diameter 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

UDP Sucrose P Synthase

Sucrose P
Sucrose P
Pi Phosphatase
Sucrose
Invertase Sucrose Synthase
UDP
Fructose Fructose
+ +
Glucose UDP-Glucose
 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

Spin at 36000g

36000g pellet 36000g

supernatant

Activity per fraction (µmol/h)

Cell fraction SPS Rubisco

2000g pellet 0.74 273 ie SPS is in the cytosol

36000g pellet 2.6 74
Supernatant 19.3 350
 Permeability of Chloroplast Envelope

• How does fixed carbon get to the cytosol?

– Uptake of labelled compounds into isolated chloroplasts

125
3 PGA CH2OP CH2OP CH2OP
| | |
hexose P
100 CHOH C=O CHOH
| | |
COOH CH2OH CHO
nmol/mg chl

75
3-P glycerate DHAP Glyceraldehyde
3-P
50

25 Triose phosphate and 3-Pglycerate can

cross envelope at rates comparable to
0 photosynthesis
0 10 20 30 40
time (sec)
 Phosphate Translocator
• Competition experiments
– uptake of 3-carbon compounds by same carrier
– strict counter exchange for Pi

150
nmol/mg chl

100

50
Pi released
3-PGA taken up
0
0 100 200 300 400
time (sec)

• 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 CH2OHO
O O
OH
Glucose 1-P
OH O-P-O-P-O-uridine
PPi OH O- O-

UDP-G
pyrophosphorylase
Pathway of sucrose synthesis from CO2

CO2

RuBP
CO2 DHAP
DHAP Ga3P
3PGA
Ga3P FBP
1,3 bisPGA Pi
F6P

G6P
sucrose sucrose P
G1P
UTP
PPi
UDGP
 Enzymes of Starch Synthesis

ATP
Glucose 1-P ADPglucose
PPi
ADPG PPiase  1,4 glucann

Starch
synthase
 1,4 glucann
Glucose 1-P  1,4 glucann+1
Pi Starch
Starch
phosphorylase
 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 -1,4 links
ADP-G
starch
synthase
ADP
The First Plant Biochemist

Gregor Mendel
round versus wrinkled peas
1822-1888
 Mendel’s Wrinkled Peas
• wrinkled (rr) peas have less amylopectin than wt - collapse
on drying
• ‘Branching Enzyme’ Activity in embryos
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

• 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

start

-1,4 link
branching enzyme

-1,6 link
 Synthesis of Transitory Starch

source cell

CO2
PGA
RuBP
1,3 bisPGA

starch Triose P

sucrose
chloroplast

export to
• ADP-G PPiase, Starch synthase phloem
and BE all found in chloroplast
 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
+ amylose + amylopectin
modified
• fried snacks starch
• Improve freeze-
(crispness / browning) thaw of frozen food
• thickener / • paper strength
• Phosphate content • adhesive
gelling agent
• water absorbency • livestock feed
• biodegradable
• improve starch addition
packing material
• film coating granule integrity
(cross linker)