plant_metab.ppt synthesis of reserves and their mobilisation

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

1. Management of Reserves
Alison Smith ([email protected])
•CO
2fixation produces triose P
•conversion to sucrose for translocation to sink tissues
•nutrient assimilation -N, S, P –and synthesis of all
cellularcomponents
•synthesis of reserves and their mobilisation
•regulation is essential

2. Management of Reserves
Lectures
9. Carbohydrate synthesis, transport and storage
10. Lipid synthesis, mobilisation of reserves
11. Regulation of metabolism

3. 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?!

4. chloroplast
Photosyntheticcell
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

5. 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

6. Sugar translocation
•Analysis of phloem sap
–shallow incision produces little sap
CH
2OH CH
2OH
| |
HCOH HOCH
| |
HOCH HOCH
| |
HCOH HCOH
| |
HCOH HCOH
| |
CH
2OH CH
2OH
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

7. 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
SucroseRaffinoseStachyose 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) ++ + ++++ -

8. 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 Pstarch
sucrose

9. 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
2assimilated 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

10. 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 µm in diameter
rice: angular,
10 µm in diameter
acceptors
for addition
of further
glucose units
start
(reducing end)
polymer of glucose units

11. 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

12. 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?

13. 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

14. •Relationship between enzyme activities and sucrose synthesis
Sucrose is made via Sucrose-P
•
14
CO
2incorporation experiments show label goes from
UDP-Glucose sucrose-P sucrose
Activity (µmol/hr per g FW)
Tissue Sucrose P Sucrose
synthasesynthase
Spinaciaoleracea leaf 25.0 0.4
Loliumtemulentum leaf 9.60.4
Pisumsativum root stele 4.626.4
Pisumsativum root cortex 1.6 5.2

15. 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 (µmol/h)
Cell fraction SPS Rubisco
2000gpellet 0.74 273
36000gpellet 2.6 74
Supernatant 19.3 350
ie SPS is in the cytosol

16. 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/mg chl
Triose phosphate and 3-Pglycerate can
cross envelope at rates comparable to
photosynthesis
CH
2OP CH
2OP CH
2OP
| | |
CHOH C=O CHOH
| | |
COOH CH
2OH CHO
3-P glycerate DHAP Glyceraldehyde
3-P

17. 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)
nmol/mg chl

18. 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
2OH
OH O-P-O-P-O-uridine
OH
OH
O
O
-
O
-
OO
Glucose 1-P
UDP-G
pyrophosphorylase
UTP
PPi

19. Pathway of sucrose synthesis from CO
2
DHAPCO
2
Ga3P
3PGA
RuBP
1,3 bisPGA
CO
2
FBP
Ga3P
sucrose P
F6P
G6P
Pi
DHAP
sucrose
UTP
PPi
G1P
UDGP

20. Enzymes of Starch Synthesis
1,4 glucan
n+1Glucose 1-P
1,4 glucan
n
Starch
phosphorylase
Pi
ADPglucose
Starch
synthase
1,4 glucan
n
Starch
Glucose 1-P
PPi
ATP
ADPG PPiase

21. 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

22. The First Plant Biochemist
Gregor Mendel
1822-1888
round versus wrinkled peas

23. •wrinkled(rr) peas have less amylopectin than wt -collapse
on drying
Mendel’s Wrinkled Peas
•One isozyme of BE has 0.8 kbp transposon in rlocus
(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

24. Branching Enzyme
•Branching enzyme forms the -1,6 links
start
start
branching enzyme
starch synthase
-1,4 link
-1,6 link

25. 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

26. 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!

27. 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