Plant Metab

8/12/2019 Plant Metab

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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


2/27

Management of ReservesLectures

9. Carbohydrate synthesis, transport and storage

10. Lipid synthesis, mobilisation of reserves

11. Regulation of metabolism


3/27


4/27

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


5/27

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


6/27

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


7/27

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) ++ + ++++ -


8/27

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


9/27

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 starchin 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


10/27

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


11/27

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


12/27

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?


13/27

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


14/27

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


15/27

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


16/27

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


17/27

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


18/27

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


19/27

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


20/27

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


21/27

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


22/27

The First Plant Biochemist

Gregor Mendel

1822-1888 round versus wrinkled peas


23/27

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


24/27

Branching Enzyme

Branching enzyme forms the -1,6 links

start

start

branching enzyme

starch synthase

-1,4 link

-1,6 link


25/27

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

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Plant Metab