Management of ReservesManagement of Reserves Alison Smith (as25@cam.ac.uk)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 Management of Reserves LecturesLectures

9. Carbohydrate synthesis, transport and storage

10. Lipid synthesis, mobilisation of reserves

11. Regulation of metabolism

Lecture 9 - Lecture 9 - Carbohydrate Synthesis, Transport and Carbohydrate Synthesis, Transport and

StorageStorage

• Sugars produced by plant

• Pathway of sugar synthesis from photosynthate

• How do you work out what’s happening?!

chloroplast

Photosynthetic cell

Synthesis of Starch and SucroseSynthesis of Starch and Sucrose

sucrose

starch

1,3 bisPGA

PGACO2

RuBP

Triose P

• Sucrose is principal photosynthetic product

– accounts for most of CO2 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-phosphoglycerate1,3 bisPGA = 1,3 bis-phosphoglycerate

Sugar Translocation is EssentialSugar 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 translocationSugar 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

Sugar composition of phloem sapSugar 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) ++ + ++++ -

Photosynthetic cell• transitory starch storage• green leaves

Starch is made in photosynthetic and non-Starch is made in photosynthetic and non-photosynthetic cellsphotosynthetic cells

amyloplast

Non-photosynthetic cell• long-term starch storage• roots, tubers, seeds

starch

chloroplast sucrose

Triose Pstarch

sucrose

Importance of StarchImportance of Starch

Sunflower after 47 min photosynthesis

Carbon absorbed (mg) 7.87Hexose accumulated 1.17Sucrose 4.20Starch 1.84

• Starch is the dominant storage polysaccharide in most plants

• In leaves - transitory starch - in chloroplasts– high percentage of CO2 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 StarchComposition 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 additionof furtherglucose units

start(reducing end)

polymer of glucose units

FructansFructans

• 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?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 MetabolismEnzymes of Sucrose Metabolism

Sucrose

UDP-Glucose Fructose 6P

Sucrose P

Pi

Sucrose P Synthase

Sucrose PPhosphatase

UDP

Fructose +UDP-Glucose

Sucrose SynthaseUDP

Fructose +Glucose

Invertase

• Relationship between enzyme activities and sucrose synthesis

Sucrose is made via Sucrose-PSucrose is made via Sucrose-P

• 14CO2 incorporation experiments show label goes from

UDP-Glucose sucrose-P sucrose

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

Location of sucrose synthesisLocation 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 (µmol/h)

Cell fraction SPS Rubisco

2000g pellet 0.74 27336000g pellet 2.6 74Supernatant 19.3 350

ie SPS is in the cytosol

Permeability of Chloroplast EnvelopePermeability 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

nm

ol/

mg

ch

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

Phosphate Translocator 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 400time (sec)

nm

ol/

mg

ch

l

Synthesis of UDP-GlucoseSynthesis 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-uridineOH

OH

O

O- O-

OOGlucose 1-P

UDP-G pyrophosphorylase

UTP

PPi

Pathway of sucrose synthesis from COPathway of sucrose synthesis from CO22

DHAPCO2

Ga3P3PGA

RuBP

1,3 bisPGA

CO2

FBP

Ga3P

sucrose P

F6P

G6P

Pi

DHAP

sucrose

UTP

PPi

G1P

UDGP

Enzymes of Starch SynthesisEnzymes of Starch Synthesis

1,4 glucann+1Glucose 1-P

1,4 glucann

Starch phosphorylase

Pi

ADPglucose

Starch synthase

1,4 glucann

Starch

Glucose 1-PPPi

ATP

ADPG PPiase

Starch Synthesis Starch Synthesis in vivoin 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

ADP-Gstarch synthase

The First Plant BiochemistThe First Plant Biochemist

Gregor Mendel1822-1888 round versus wrinkled peas

• wrinkled (rr) peas have less amylopectin than wt - collapse on drying

Mendel’s Wrinkled PeasMendel’s Wrinkled Peas

• One isozyme of BE has 0.8 kbp transposon in r locus (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

Branching EnzymeBranching 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 StarchSynthesis 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

CO2

RuBP

Lecture 9 - SummaryLecture 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 Genetic or biochemical modifications of starch are or may be used for...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