Topic:

Engineering tobacco plants for enhanced stress tolerance:

The importance of primary metabolism VL I

Prof. Dr. Antje von Schaewen„Molecular Physiology of Plants“University of Münster, Germanyschaewen@uni‐muenster.de

Plants are sessile organisms

?!

endogenous cues(development)

exogenous cues(abiotic & biotic stress)

Environment(Light, water, solutes / salt, temperature)

Friends / Foes (symbiotic versus pathogenic bacteria or fungi, viruses)

Intra-cellular signaling networksadequate responses

Altered CHO partitioning in leaves affectssink-to-source transitions

Secretion of yeast invertase into the apoplastSNN::P35S-secSUC2-OCSpA

Photosynthesis too close to optimum

von Schaewen A, Stitt M, Schmidt R, Willmitzer L (1990). EMBO J. 9: 3033-3044. Stitt M, von Schaewen A, Willmitzer L (1990). Planta 183: 40-50. Sonnewald U, Brauer M, von Schaewen A, Stitt M, Willmitzer L (1991). Plant J. 1: 95-106.

PSstimulation

soluble sugars

phot

osyn

thet

ic(P

S) c

apac

ity

Optimum

PS decline

Celldeath

PSstimulation

soluble sugars

phot

osyn

thet

ic(P

S) c

apac

ity

Optimum

PS decline

Celldeath

A41-10WT

sucrose Glc Fru

invertase

Sites of ROS production & scavenging in plant cells

H2O2

Chloroplast

NADP+Cytosol

NADPH

Peroxisome

Mito

NADPHH2O2

H2O2

NAD(P)H

NADH

„Mehler reaction“

H2O2

„oxidative burst“ (reactive oxygenspecies, ROS)

„Photo-respiration“

„Respiration“

Cell wall

AvS (2005)

PSIPSII

[GSH]2

[GSSG]

O2-

Rboh

Glutathion(-L-Glutamyl-L-cysteinyl-glycin) a peptide made of 3 amino acids

Glutaminic acid (Glu, E) Cystein (Cys, C) Glycin (Gly, G)

E-C-GSH

E-C-G

Main redox bufferin most cellular compartments(and Cystein store)

SH

2 GSH

E-C-GS

E-C-GS

GSSG

2 H

Antioxidant

inter-molecular disulfide!

Trxred

targetox

-SH-SH S-

Trxox

targetred

HS-HS-

-S-S

red/ox switch

Thioredoxin

S-

Dithiothreitol (DTTred) = Cleland‘s reagent

Sulfated derivatives of sugar alcohol threitol, serving as antioxidant

D L

H2C-SH

C-SHH2

H HO-C-

HO-C-H

2 H

Air (O2)

H2C-S

C-SH2

H HO-C-

HO-C-H

L and D are stereo-isomeric forms

DTTred DTTox

intra-molecular disulfide!

Intracellular redox (signaling?) networks

NADPH

NADP

PRX

2 R-SH + H2O2→ 2 R-SOH + H2O

[R-SO2H = inactive]

PRX = Peroxiredoxins(pla. 2-Cys-Peroxidases)

[GSH]2

[GSSG]

GRX

Target enzymemodulation

(roGFP)

GRX = Glutaredoxins

GR

GPX GPX = Glutathion-

Peroxidases(Seleno-Cys in active centre in

animals)

R-H2 + H2O2→ R‘ + 2 H2O

Glutathion = main redox buffer in most subcellular compartments

„Halliwell-Asada-Foyer“

TRX

Target enzymemodulation

FNR,Fd/FTR

TRX = Thioredoxins

(in the cytosoland insideplastids)

?

Light/dark-dependent redox regulation of RPPP and OPPP enzymes in the chloroplast stroma prevents futile cycling

CO2

ATP

2 NADPH2 ATP

Export of Triose-Phosphates (GAP

and DHAP)

NADPH

NADPH

G6PDH

2x2x

2x

Pi

Pi

NADP

NADP

2 NADP

ADP + Pi

2 ADP

Michels & Wedel (2001)Geigenberger (2011)

PRK

GAPDH

FBPase

SBPase

starchAGPase

CO2

SH

SH

S

S

Disulfide-/dithiol interchange

H2O 1/2 O2

Light (Fd red)2 e- + 2 H+

EE

Tdox

Tdred

Post-translational enzyme regulation

De-/Phosphorylation

E

ATP ADP

H2OPi

Kinase

Phosphatase

EP

Soluble sugars, redox state &Glucose-6 phosphate dehydrogenase (G6PDH)

Initial enzyme of the OPPP (= oxidative pentose-phosphate pathway)Alternative C6 breakdown (primary metabolism) Provides NADPH for anabolic biosyntheses (amino acids, etc.)

In plants, G6PDH isoenzymes are present in the Cytosol (cyt) and in Plastids(P1, P2 as formal inversion of the Calvin cycle = reductive pentose-P pathway)

6PGDH Ru5P6PGG6P PGL

NADPHNADPH

CO2

Lacto-nase

G6PDH

Chloroplaststarch

Role of GAPN(= non-phosphorylating GAPDH)

Indirect Export of NADPH -from the chloroplast stromato the cytosol - in the light (Kelly and Gibbs, 1973)

3PGA

NADPN

G6P

RPPP (Calvin cycle)

CO2

TPT Ru5P

F6P

ATP

NADPH

NADPH

Triose-P

Situation in the light…

lightATP

GAPN = alternative NADPH source at C3 level in the cytosol.

P1 G6PDH „off“(and OPPP)

Triose-P

3PGA

Peroxi-some?

CO2

Ru5P

Citrate cycleMito-chondrium

Cytosol Sucrose

Gluco-neogenesis

3PGA

PEPGlycolysis

ATPPyr

F6P

F1,6P2

NADPH

NADP

ADP (+Pi)+ NAD

NADH

G6P

Chloroplast

Pi

Pi

H2O

Mal +

Glc

Glc

PEP

Fatty acid synthesis

E4P

Shikimate pathway

Pi

Phenylpropanes

Chorismate

G6P

Pi

OPPP

P1

CO2

NADP

C7 +Triose-P

Pi

Ru5P

PPT

TPT

GlcT

Nucleotidesynthesis

NADPH

starch

XPT

Glyco-lysis

Situation in darkness…

NADP

NADPHN

cyt

C

AB

GAPD isoforms

GAP

G6PD

G6PD isoform?

Triose-P NAD +

NADP binding domain

G6P binding domain

Role of cysteine residues in plastidial G6PDH enzymes

Identification of redox-active Cys in St P1: Wenderoth et al. (1997) J. Biol. Chem. 272: 26985-26990.Isolation of St P1 G6PD isoform: von Schaewen et al. (1995) Plant Physiol. 109: 1327-1335.Isolation of St cyt G6PD isoform: Graeve et al. (1994) Plant J. 5: 353-361.

C11

9

C14

9C

157

C16

8

C19

4C

216

mature proteinTP

cyt G6PDH activity is not influenced by redox changes

C6

C29

C15

5C

162

C27

6

C37

9

Cyano

Cyt

C18

9

C47

7

P1

*redox relevant, Cys Ser mutations *

with isoform-specific

cDNA probes

with isoform-specific

antisera

in oxidised & reduced state

(-/+ DTTred)

RNA isolationNorthern-Blot

Protein isolationWestern-Blot

MeasureEnzyme activities

6 h bis 72 h incubation on different solutions(e.g. water, sugars, or Paraquat in the dark)

Extraction

Wildtype potato plants (4 to 6 weeks old)

Qestion: How much „cross talk“ between plastids & cytosol is there?

Leaf incubation experiments

Sugar feeding stimulates mRNA expression and increased activity of cytosolic G6PDH

0

100

200

300

400

500

0 12 24 36 48

H2O

Glc

Fru

Suc

Act

ivity

pers

urfa

ce(U

·m-2

)

Incubation time (h)

0

100

200

300

400

500

0 12 24 36 48

H2O

Glc

Fru

Suc

Act

ivity

pers

urfa

ce(U

·m-2

)

Incubation time (h)

H2O Glc

0 12 24 48 12 24 48

1.8 kb

The DTTred-sensitive activity (plastidic G6PDH isoenzymes) did not change – what is DTT and what exactly does that mean?

cytosolic G6PDH activity (DTTred-insensitive)mRNA

cyt G6PDH

Inactivation of ROS in chloroplasts (dark transition)

.

ASA

DHA

GSSG

GSH

GSHRDHAR

APX

MDARMDA•¯

„Halliwell-Asada“-Weg .

NADP+ NADPH

OPPP

6PGDH

G6P

Ru5P

SH SHG6PDH

Fd

PS I

.

Nitrogen assimilation

Sulfur assimilation

Stroma

Thylacoid lumen

FNR

e-

NADP+

Pq

2 O2 + 2 H2ONFR

Calvin cycle (RPPP)

2 H2O + O2[4 H+]

ASA

DHA

GSSG

GSH

GRDHAR

APX

MDAR

•MDA¯

„Halliwell-Asada-Foyer“ pathway .

•OH¯

„Haber-Weiss“

•O2¯ H2O2

SODO2

„Mehler reaction“

S—S

PS II

Troll

0

10

20

30

40

50

60

0 5 10 15 20 25 30 35 40 45

0

10

20

30

40

50

60

0 5 10 15 20 25 30 35 40 45

PQ

Glc + SO4-

SO4-

H2O

PQ

Glc + NO3-

NO3-

H2O

Paraquat (PQ) data

DTT-sensitive = chl. G6PDH activity

The DTT-insensitive G6PDH activity (of cytosolic isoenzymes) did not change, and neither mRNA nor protein levels of the chloroplast G6PD isoform.

chl. G6PDH is phosphorylated (in the dark, D)

Immuno-precip. of 32P-labeled protein

De-phosphorylation may explain 10foldactivation of the oxidized enzyme

Model of post-translational G6PDH regulation in chloroplasts

Dark Light

„off“

SH

G6P

HS

G6PDHred.

ox.

Stroma

KinasePhosphatase

maximal„on“

minimal„on“

P

G6P

G6PG6PDH

G6PDH

Hauschild & von Schaewen (2003) Plant Physiol. 133: 47-62.

Hauschild & von Schaewen (2003) Plant Physiol. 133: 47-62.

Nucleus

Cytosol

SURE ATG cyt G6PD-1800 -700

mRNA

Chloroplastsugar sugar

sensor

G6P

C5P

NADPH

NADPH+ CO2

6PG

NADP

G6PDH

sugar

ChxOPPP

C5P

G6P

6PG

light

redoxsensor

E4P

NADPHNADP

Pi

NADPH

NADPH+ CO2

NADPG6PDH

starchATP

PQ ROS

DCMU

cytG6PD antisense potato lants show no phenotype

WT P1 P80 WT P1 P80

Leaf Tuber

Northern Blot (DIG-system)

0,1 ng

1 ng

100 ng

10 ng

0,01 ng

Cyt P2 P1Antisense linescytG6PD is strongly suppressed in tubers, but lesscomplete in leaves of P1 and P80 transformants.

Potato lines of Kerstin Graeve (1994)

WT P1 P80

source sink

Gerrit Boemke (2004)

35S P OCS pA

cyt G6PD

mature proteinTP

Wendt et al. (2000) Plant J. 23(6): 723-733.

P2C C

cyt G6PD

35S P OCS pA

Hints for presence of a 2nd plastidic G6PD isoform (P2)

( RT-PCR from P80 roots)

KiNADPH > KmNADP TP-P2-GFP

Leaf

Enz

yme

activ

ity (m

U m

g-2 ) 20

cyt

WT P80

pla

10

Root200

cyt

WT P80

pla ?

100

Model of G6PD isoform evolution in higher plants

Wendt et al. (1999)Plant Mol. Biol. 40: 487-494.

heterotrophic plastids

SSP2

Nucleus

Eu-bacterium

Mito

Chloroplasts

SHSH

Fd

NADPH

P1

Cya

G6PD mRNA species

P1 SHSH

MitoP2

Cyt

SHSH

NADPH

FdGOGAT

NiR

gene transfer

Cyt

gene duplication+

modification

Cytosol