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