Glutathione S-transferase isoenzymes in plant defense reactions against stress and disease

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Glutathione S-transferase isoenzymes in plant
defense reactions against stress and disease

• Gábor Gullner
• Plant Protection Institute
• Hungarian Academy of Sciences
• Budapest, Hungary

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Glutathione S-transferase (GST) activity first
detected in maize
Frear and Swanson, 1970
glutathione
HCl
atrazine
conjugate
High GST activity maize, sorghum
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Glutathione (GSH)
Cys
Gly
Glu
SH
?-L-glutamyl-L-cysteinyl-glycine
- most important non-protein thiol compound
1 mM in cytosol - very reactive sulfhydryl
group - detoxification reactions - antioxidant
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Several herbicides are rapidly metabolized
via glutathione conjugation in plants. -
different GST isoenzymes exist in plants
Often GSTs are the basis of herbicide
selectivity - crop plants (maize) high GST
activity - competing weeds low GST activity
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Herbicide detoxification by GSTs
Triazines atrazine, simazine Chloroacetanilides
acetochlor, alachlor, metolachlor Thiocarbamate
s EPTC Nitrodiphenylethers acifluorfen,
fluorodifen Sulfonylureas chlorimuron
ethyl Others chlorfenprop-methyl, metribuzin
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Transgenic tobacco
Transformation of tobacco with a maize
gene encoding the GST27 isoenzyme
conferred tolerance to chloroacetanilide
herbicides (Jepson et al., 1997)
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Herbicide safeners (antidotes)
Wide range of synthetic compounds They increase
the herbicide tolerance in crop plants (usually
in cereals) Mode of action Selective induction
of GST isoenzymes participating in herbicide
detoxification
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Other toxic GST substrates
1,2-dichloro-4-nitrobenzene 1-chloro-2,4-dinitrob
enzene 4-nitropyridine-N-oxide p-nitrophenethyl-
bromide p-nitrobenzyl chloride bromosulfophthale
in
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Classical definition of GSTs GSTs catalyze the
conjugation of glutathione to a wide range of
toxic and hydrophobic compounds large isoenzyme
family predominantly cytosolic in maize 1-2 of
all soluble proteins endogenous, physiological
substrates??
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Transport to the vacuoles
ATP-dependent membrane pumps recognize
the glutathione S-conjugates and transfer them
into the vacuoles Glutathione conjugation tags
the xenobiotic substrates for recognition by
membrane pumps
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Measurement of total glutathione S-transferase
(GST) activityspectrophotometrically at 340 nm
conjugate
CDNB
Best model substrate 1-chloro-2,4-dinitrobenzene
(CDNB)
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Separation of GST isoenzymes by HPLC in wheat
extracts
Crude extract (NH4)2SO4 precipitation affinity
chromatography on GSH-agarose column RP-HPLC UV
detection (214 nm)
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Old history (1990s) GST isoenzymes in maize
Two subunits Homo- and heterodimer molecules co
nstitutive or inducible
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GSTs today
Maize 42 isoenzymes EST (expressed sequence
tag) database BLAST searching (Du Pont Company,
USA) Arabidopsis thaliana 47 isoenzymes McGonig
le et al., Plant Physiol. 124, 1105-1120 (2000)
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Classification of GST isoenzymes
Based on intron-exon gene structure
amino acid sequence similarity
immunological cross reactivity
Phi class 2 introns, major maize GSTs Zeta
class 9 introns Tau class 1 intron, auxin
inducible Theta class 6 introns
Edwards et al., 2000
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GSTs in endogenous metabolism
GSTs are not only stress- and detoxification
enzymes Other activities GST has high affinity
for auxins and cytokinins ligandin function,
hormone homeostasis (storage)? auxin carrier,
transport between cell compartments anthocyanin
sequestration in the vacuoles
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Structure of GST proteins
X-ray crystallography Arabidopsis Phi class
GST Two subunits, both with two distinct
domains Active site G-site conserved GSH
binding site (in N terminal domain - in
red) H-site binding pocket for hydrophobic
cosubstrates - serine residue activates GSH for
conjugation
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Inducibility of GSTs
H2O2 and other ROS xenobiotics safeners heavy
metals cold, heat drought plant hormones pathogen
infections salicylic acid (SA)
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Peroxidase activity of GST
R-OOH 2 GSH ? R-OH GS-SG H2O
Detoxification of organic hydroperoxides Toleranc
e against oxidative stress Hydrogen peroxide is
not a substrate Glutathione peroxidase -
different enzyme
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GST activity in cold-hardened cereals
Cold-hardening gradually decreasing temperatures
for 7 weeks between 14 and 3 ?C ? increasing
frost resistance Control 17 ?C
Strong induction But no correlation between
GST induction and frost tolerance
Janda et al., Plant Science 164, 301-6 (2003)
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Futile gene induction?
Ozone fumigation of tobacco caused a strong
induction of basic ?-1,3-glucanase and chitinase
activities and mRNA levels Pathogenesis-related
proteins Function?? Sandermann et al., 1996
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Tobacco overexpressing GSTincreased stress
tolerance
Transformation Chimeric Nt 107 gene encoding GST
under control of an enhanced CaMV
promoter Increased stress tolerance against -
salt stress - chilling stress Reduced lipid
peroxidation Enhanced growth of seedlings during
stress
Roxas et al., 2000
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Injection of salicylic acid (0,8 mM) into leaves
of Xanthi-nc tobacco
of control
GST activity
Days after treatment
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Induction of GSTs by salicylic acid (SA)
SA-responsive element in the promoter of several
GSTs activation sequence-1
(as-1) approx. 20 base pairs, contains two TGACG
motifs that bind basic/Leu zipper transcription
factors
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Salicylic acid auxin methyl
jasmonate
reactive oxygen species (ROS)
as-1 binds nuclear transcription factors
GST induction
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Induction of GST genesafter oxidative stress
oxidative stress response element coding
region
DNA-protein binding
nuclear factor
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Activation of nuclear transcription factors
phosphorylation
OH
O P
ROS
inactive protein active
protein
binding to as-1
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Activation of nuclear transcription factors
disulfide bridge formation
SH SH
S S
ROS
inactive protein active
protein
binding to as-1
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Similar factors in animal cells
nuclear factor-?B AP-1 transcription factor
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Detection of DNA-protein binding gel mobility
shift assay
1. isolation of nuclear proteins 2. incubation
with 32P-labeled DNA probe containing
promoter element 3. polyacrylamide gel
electrophoresis of DNA probes with and
without nuclear proteins 4. autoradiography
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Activation of as-1 element
in a transgenic tobacco construct
1 mM SA, light ? accumulation of ROS ? as-1
promoter element can bind nuclear
protein Antioxidants inhibit the SA induced gene
expression
(Hidalgo et al., 2001 Garretón et a., 2002)
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GUS reporter gene
?-glucuronidase hydrolyzes a wide range of
?-glucuronides Widely used in histochemical
studies Low endogenous activity in
plants Substrate X-Gluc 5-bromo-4-chloro-3-indo
lyl-?-glucuronide Cleavage, oxidation of indole
derivative ? dimerization ? formation of
insoluble indigo dye
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Glutathione S-transferase (GST) in wheat after
fungal infection
Dudler et al., 1991
Winter wheat was infected by barley
powdery mildew (Blumeria graminis f.sp. hordei)
Resistance against wheat powdery
mildew Accumulation of mRNA encoding GST (29 kD
protein) This GST was specifically induced by
infection Its role ??
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GST inductions in plant-pathogen interactions
Hahn és Strittmatter, 1994
Potato leaf - fungus (Phytophthora infestans)
Greenberg et al., 1994 Ádám et al., 1997
Bean, Arabidopsis - Pseudomonas syringae pv.
phaseolicola
Venisse és mtsai., 2001
Tobacco, pear - Erwinia amylovora
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Xanthi-nc tobacco infected with tobacco mosaic
virus (TMV)
incompatible interaction
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Induction of GST activity in tobaccoleaves
infected with tobacco mosaic virus
Spectrophotometric analysis (340 nm) - total GST
activity
Fodor et al., Plant Physiol. 114, 1443-1451 (1997)
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Induction of GST by the monoterpene oil S-carvone
in Xanthi-nc tobacco leaf discs
GST activity

caraway, dill
Days after treatment
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Visible symptoms of TMV infection in Xanthi-nc
tobacco leaf discs after (S)-carvone pretreatment
30
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Number of lesions
20
15

10
5
0
TMV (S)-carvone

TMV
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Virus concentrationswith and without
(S)-carvonepretreatment by ELISA test
140
120
arbitrary units
100
80
60
40
20
0
TMV (S)-carvone
TMV
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Effect of GST on the number of necrotic lesions
Wagner and Mauch, 2000
Xanthi-nc tobacco plants were transformed by a
wheat GST gene, which can be induced by fungal
infection - CaMV 35S promoter
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Glutatione S-transferase isoenzymes are able to
suppress the development of necrotic lesions by
an unknown mechanism.
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TMV infection
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The role of glutathione S-transferase isoenzymes
in infected plants is the detoxification of toxic
lipid hydroperoxides, which are produced during
lipid peroxidation.
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Future plans Study of the induction of
specific GST isoenzyme(s) in TMV infected
tobacco leaves on mRNA level by
RT-PCR RT-PCR excellent tool to study gene
inductions - isolation of total mRNA from virus
infected leaves - synthesis of cDNAs by reverse
transcriptase - PCR with GST specific or
degenerate primers - sequencing - cloning into
bacteria, functional studies
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Plant Protection Institute, Budapest