Ranunculus ficaria

Starch Biosynthesis and Improved Starch Production

Starch Biosynthesis and Improved starch production

1.1 Properties of starch

1.2 Carbon flow during starch synthesis

1.2.1 Enzymes and intermediates

1.2.2 Regulation of starch synthesis and of amylose/amylopectin ratio

1. Starch

2. Improved starch production

Glycogen

- All animals (e.g. human liver) many microbes

- Carbohydrate storage

- Highly branched (high solubility)

- UDPGluc serves as activated sugar

- Glycogenine (protein, that (auto)catalyzes a first Glc moiety from UDPGlc to Tyr194)

- Next reaction catalyzed by Glycogen Synthase)

UDP-Glc + Tyr194-Glc

UDP + Tyr194-Glc-Glc

Glycogen Synthase is covalently modified

GS + ATP GS-P + ADP aktiv inaktiv

Starch is closely related to glycogen

Glycogen Synthase

Starch: different sources, different applications

assembled (zusammengesetzt) starch

Starch kernels appear in individual shapes

reducing end

Amylopectin: 500000 - mill Glc units

every 15 - 30 α1.4-linkage one additional α1.6-linkage

Starch consists of two types of molecules: amylose and amylopectin

Amylose: 3-7000 Glc units, form α-helical structure (6 Glc residues per loop)

Amylose is α-helical organized

Starch test: J2 is de facto a proof for amylose not for starch in total

starch free zones cor-respond to chlorophyll free zones

Spherical structure of starch is determined by amylopectin

amorphous lamellae of loose branching

crystalline lamellae of dense helical arrangement

mut

Wt

Starch metabolism must be regulated !

Characteristics of starch less mutants 1. increased shoot/root ratio

2. low tolerance against drought stress

3. grow only at optimal nutrient conditions

4. increased respiration

5. normal phenotype under permanent light

starch less

wild type

starch less wild type

In some algae starch synthesis takes place outside the plastid

nucleo- morph

- schematic -

Starch

nucleus

periplastidial space

plastid

pyrenoid

cER

cytosol

-TEM -

pps

starch

Guillardia theta

Guillardia starch kernels show a unique shape (inner cavity)

Starch biosynthesis starts with hexose phosphates

AGPGlc pyrophophorylase (AGPase) is the “rate limiting” step

- rate limiting step

- immediate hydrolysis of PPi - in plants heterotetramer

2 α-UE (50 kDa), 2 β-UE (53 kDA)

- allosteric effectors Pi und 3-PGA

- 3-PGA/Pi ratio is critical for resulting V

+ -

Three levels of AGPase regulation

- +

Starch synthases produce the glucose polymer

in addition: individual RNA processing and first evidence for post-transcriptional modification by phosphorylation leads to individual starch synthase enzymes

two isoenzymes of Starch Synthases: soluble Starch Synthase SSS granula-bound Starch Synthase GBSS SSS: amylopectin synthesis (high substrate affinity) GBSS: amylose synthesis (low affinity, located on and in kernels)

We always find isoforms of Starch Synthases !

Intra-transfer (within a chain) Inter-transfer (two chains involved)

OH

OH

OH OH OH

OH

OH

Residual segment

Acceptor-chain

Donor-chain

Branching Enzymes produce α1.6-linkages

OH

Branching Enzymes

Start

We keep in mind:

Starch-free mutants show substantial developmental defects

Starch consists of amylose and amylopectin; in higher plants always in plastids

AGPase is regulated allosterically and by covalent modification. 3-PGA is activator; Pi acts as an inhibitor. Reduced thioredoxin activates AGPase in addition (AGPasered). High cellular sugar levels promote expression of AGPase genes

Regulation of starch biosynthesis occurs on the level of ADPGlucose Pyrophosphorylase (rate limiting step)

Improved Starch Metabolism in Higher Plants

ADP

Mitochondrion Amyloplast

starch fatty acids

2 x

ATP ATP

ADP ADP

Tjaden et al., 1998a; Trentmann et al., 2000

Nucleotide transport in potato tuber cells

Adenosine triphosphate (ATP)

Nucleotide transport

DNA / RNA Enzyme cofactors

Second Messenger Precursors for hormones

universial energy currency

Nucleotides, the most important metabolites on earth !?

TWINLAB

80 mg/day 30 kg/day

demand

Nucleotides, the most important metabolites on earth !?

Starch synthesis in potato tubers

ATP ATP

STARCH

ADPGlc

Glc1P

Glc6P

AGPase ATP

ADP

ATP ADP

Pi

potato amyloplast

Glc6P

x

ginger-like morphology

budding tubers

high sugar, low starch content iodide staining

first step of starch synthesis

Wild type NTT antisense

NTT activity is crucial for starch biosynthesis; but does it limit starch levels ?

WT NTT antisense lines

Tjaden et al., 1998 Plant J. 16

Tubers from antisense lines destain easily after iodine treatment, WT tubers do not!

Wild type NTT antisense

Iodine staining

Wild type NTT antisense

line1 NTT antisense

line1

Iodine de-staining

30 min, hot water triiodite captured in amylose

Tjaden et al., 1998 Plant J. 16

Amylose structure

Amylose, α-1.4 linked, linear glucose polymer

- Up to 1000 glc units/molecule

Amylopectin structure

Amylopectin

Amylopectin, α-1.4 and α-1.6 branched glucose polymer

- every 15-30 α-1.4 linkages a branch at position 1.6, up to 105 glc units

Carbon pathway into the two products: amylose and amylopectin

GBSS, sticks to starch granule surface

SSS, soluble in stroma

low ADPGlc affinity !

high ADPGlc affinity !

Antisense lines show low starch, low amylose but high sugar levels Tjaden et al., 1998 Plant J. 16

triiodite captured in amylose

first step of starch synthesis

Glc1P ADPGlc

ATPATP

STARCH

ADPGlc

Glc1P

Glc6P

AGPase ATP

ADP

ATP ADP

Pi

potato amyloplast

Glc6P

Can we increase starch production by increased provision of Glc6P or ATP

GPT2 mRNA NTT mRNA

Overexpression of GPT or NTT alone does not improve potato starch levels

Zhang et al. 2006, Plant Biotech. J. 6

NTT mRNA on basis of GPT

overexpressors

Simultaneous Overexpression of GPT or NTT improve potato starch levels

Zhang et al. 2006, Plant Biotech. J. 6

Zhang et al. 2006, Plant Biotech. J. 6

As a consequence, overexpressors contain more amylose

How to produce very-high amylose potatoes ?

GBSS, sticks to starch corn surface

SSS, soluble in stroma

Western-blot analysis revealed massively reduced SBE-A and SBE-B levels

Schwall et al, 2000, Nat.Biotech. 18

Production of very-high-amylose potato starch by inhibition of SBE A and B

Schwall et al, 2000, Nat.Biotech. 18

Production of very-high-amylose potato starch by inhibition of SBE A and B

no change in total yield

Hofander et al, 2004 Plant Biotech. J. 2

Wt SBE antisense

Production of very-high-amylose potato starch by inhibition of SBE A and B

polarized light

Schwall et al, 2000, Nat.Biotech. 18

Hofander et al, 2004 Plant Biotech. J. 2

Wt SBE antisense

iodine staining

iodine trapped in amylose

Carbon pathway into the two products: Amylose and Amylopectin

GBSS, sticks to starch corn surface

SSS, soluble in stroma

Visser et al. 1991 Mol. Gen. Genet. 225

Production of very-high-amylopectin potato starch (AMFLORA®) by inhibition of GBSS

AMFLORA®, BASF potato

WT GBSS antisense

WT

GBSS antisense

No staining by iodine demonstrates absence of amylose !

Visser et al. 1991 Mol. Gen. Genet. 225

Production of very-high-amylopectin potato starch by inhibition of GBSS

low GBSS activity correlates with almost complete absence of amylose !

high GBSS activity correlates with high amylose contents!

GBSS, Western-blot analysis

wild type maize low starch, brittle1 mutation

Brittle1, a member of the mitochondrial carrier family (MCF)

brittle: dürr, brüchig

Starch synthesis in monocotyledonous endosperm tissue

ADPGlc ADPGlc

STARCH

Brittle-1

cytosol

amyloplast

Zmbt1 Glc1P

AGPase

Kirchberger et al. (2007) J. Biol. Chem. 282

Effector (5x) ZmBT1

ADPG uptake

none 100 ATP 71 ADP 15

ADP-Glc 24

AMP 82

ADPGlc

ADP

antiport

ZmBt1 transports ADPGlc in an antiport mode with ADP

% of control

monocot´s possess brittle1 proteins for import of ADPGlc into the amyloplast

!

! !

Kirchberger et al. (2007) J. Biol. Chem. 282

Starch synthesis in dicot´s and monocot´s (cereals) differs

Brittle-1

ADPGlc ADPGlc

STARCH

ADP ADP

monocotyledonous amyloplast

Glc1P

AGPase

ATP ATP

STARCH

ADPGlc

Glc1P

Glc6P

AGPase ATP

ADP

ATP ADP

Pi

dicotyledonous amyloplast

Glc6P

Improvement of starch levels in maize Wang et al., 2007, Plant Cell Tissue Cult. 88, 83-92

Expression of a bacterial (E.coli) AGPase (glgC16) in maize

AGPase, ADP glucose pyrophosphorylase

Improvement of starch levels in maize. glgC16 is not Pi inhibited! Wang et al., 2007, Plant Cell Tissue Cult. 88, 83-92

The bacterial (E.coli) AGPase (glgC16) is not phosphate inhibited AGPase

AGPase, ADP glucose pyrophosphorylase

Pi

Wt AGPase inhibition by Pi

Improvement of starch levels in maize. Correlation AGPase activity/seed weight Wang et al., 2007, Plant Cell Tissue Cult. 88, 83-92

Wt seed weight