Two countries, one goal, joint success!
Antioxidant activities and reactive oxygen species in
flag leaves of Fusarium-inoculated wheat genotypes
Szeged – Timişoara axis for the safe food and feed
SZETISA1
HURO/0901/147/2.2.2
Reporting period: 01.06. 2011 – 30.09. 2011
Ágnes Gallé
Department of Plant Biology
University of Szeged
www.huro-cbc.eu
www.hungary-romania-cbc.eu
Disclaimer
The content of this page does not
necessarily represent the official
position of the European Union.
2nd Progress Report Meeting
Szeged, 22-23 September, 2011
Fusarium head blight (FHB) is an important disease of wheat and
has worldwide distribution, which results in yield loss and
mycotoxin contamination of grain when the disease is caused by a
mycotoxin-producing Fusarium species.
Fusarium graminearum and F. culmorum are the Fusarium species
that most commonly cause FHB of wheat and both can produce
trichothecene mycotoxins, predominantly deoxynivalenol (DON,
also known as vomitoxin), zearalenone, nivalenol,
dideoxynivalenol, 3-acetyldeoxynivalenol
Fusarium graminearum
Fusarium culmorum
Vomitoxin, also known as deoxynivalenol (DON), is a type B
trichothecene, an epoxy-sesquiterpeoid.
When compared to other trichothecene mycotoxins which can form in
grains and forages, vomitoxin is relatively mild.
Vomitoxin belongs to a class of mycotoxins which are strong protein
inhibitors. Inhibition of protein synthesis following exposure to
vomitoxin causes the brain to increase its uptake of the amino acid
tryptophan and, in turn, its synthesis of serotonin. Increased levels of
serotonin are believed to be responsible for the anorexic effects of
DON and other tricothecenes.
Irritation of the gastrointestinal tract may also play a role in reducing
feed intake.
Zearalenone is also a trichothecene mycotoxin, and has estrogen
effect, and can cause inflame in the uterus in swine.
Nivalenol was shown to inhibit protein synthesis in rabbit
reticulocytes in vitro, with an ID50 of 2.5 mg/ml, probably
by impairment of the ribosomal function.
Nivalenol inhibited the synthesis of nucleic acids in vitro
(Ueno and Fukushima, 1968). This response appeared at
doses much higher than the inhibition of protein synthesis.
The mechanism of nucleic acid inhibition is not known.
Plant material
GK Mini Manó/Nobeokabozu genetic mapping population
(Prof. Mesterházy Ákos )
GK Mini Manó
Arthur 71 / Sava // Rusalka / NS
171.2 /3/ F 30.74
Resistant line to e.g. leaf rust
Nobeokabozu: FHB resistant line
A recombinant line from China: ‘WSY’,
Wangshubai/Sumai3/Yangangfanzhou
inoculation
anthesis
SAMPLING
Premilk stage
Before inoculation
sampling
(ROS and total antioxidant
capacity)
Milk stage
Dough stage
Total intracellular ROS after inoculation
Total intracellular ROS before inoculation
900
900
800
700
DCF fluorescence
700
600
500
400
300
600
500
400
300
200
200
100
100
0
0
501 r
483 s
561 r
488 s
474 r
501 r
483 s
488 s
Total intracellular ROS after inoculation with F.c.
900
800
700
DFC fluorescence
474 r
600
500
400
300
200
100
0
561 r
474 r
501 r
483 s
488 s
Total intracellular ROS after inoculation with F.g.
900
800
DFC fluorescence
DCF fluorescence
800
700
600
500
400
300
200
100
0
561 r
474 r
501 r
483 s
488 s
Antioxidant activity (FRAP)
8000
Antioxidant activity
(mmol FeSO4/ g FW)
7000
6000
5000
4000
3000
2000
1000
0
561 r
474 r
501 r
483 s
488 s
Antioxidant activity (FRAP)
After inoculation with F. c.
Antioxidant activity (FRAP)
Before inoculation
8000
Antioxidant activity
(mmol FeSO4/ g FW)
7000
5000
4000
3000
2000
1000
0
6000
5000
4000
3000
2000
1000
0
474 r
501 r
483 s
488 s
561 r
474 r
501 r
483 s
488 s
Antioxidant activity (FRAP)
After inoculation with F. g.
8000
7000
Antioxidant activity
(mmol FeSO4/ g FW)
Antioxidant activity
(m mol FeSO4/ g FW)
6000
6000
5000
4000
3000
2000
1000
0
561 r
474 r
501 r
483 s
488 s
Enzymatic antioxidants
Peroxidases (POX; EC 1.11.1.7) are oxido-reductive enzymes that
participate in the wall-building processes such as oxidation of phenols,
suberization, and lignification of host plant cells during the defense
reaction against pathogenic agents.
Accumulation of lignin and phenolic compounds have been correlated
with disease resistance in a number of plant–pathogen interactions. These
include wheat, tomato and rice.
Enhanced POX activity has been correlated with resistance in rice, wheat,
barley and sugarcane following the inoculation with phytopathogens.
POX activity was rather delayed or remained unchanged during the
compatible interaction in susceptible plants. .
Peroxidase specific activity in heads of wheat cultivars. C, non-inoculated
control; I, inoculated plants. (F. graminearum)
Specific POD activity (U/ mg
protein)
POD activity of the selected w heat lines
3000
2500
2000
1500
1000
500
0
561 r
474 r
501 r
483 s
488 s
Specific POD activity (U/ mg
protein)
POD activity of the selected w heat lines after F.
culm orum infection
3000
2500
2000
1500
1000
500
0
561 r
474 r
501 r
483 s
488 s
Specific POD activity (U/ mg
protein)
POD activity of the selected w heat lines after F.
gram inearum infection
3000
2500
2000
1500
1000
500
0
561 r
474 r
501 r
483 s
488 s
Specific SOD activity (U/ mg
protein)
SOD activity of the selected w heat lines
14
12
10
8
6
4
2
0
561 r
474 r
501 r
483 s
488 s
Specific SOD activity (U/ mg
protein)
SOD activity of the selected w heat lines after F.
culm orum infection
14
12
10
8
6
4
2
0
561 r
474 r
501 r
483 s
488 s
Specific SOD activity (U/ mg
protein)
SOD activity of the selected w heat lines after F.
gram inearum infection
14
12
10
8
6
4
2
0
561 r
474 r
501 r
483 s
488 s
Specific KAT activity (U/ mg
protein)
KAT activity of the selected w heat lines
600
500
400
300
200
100
0
561 r
474 r
501 r
483 s
488 s
Specific KAT activity (U/ mg
protein)
KAT activity of the selected w heat lines after F.
culm orum infection
600
500
400
300
200
100
0
561 r
474 r
501 r
483 s
488 s
Specific KAT activity (U/ mg
protein)
KAT activity of the selected w heat lines after F.
gram inearum infection
600
500
400
300
200
100
0
561 r
474 r
501 r
483 s
488 s
Specific GR activity (U/ mg
protein)
GR activity of the selected w heat lines
600
500
400
300
200
100
0
561 r
474 r
501 r
483 s
488 s
Specific GR activity (U/ mg
protein)
GR activity of the selected w heat lines after F.
culm orum infection
600
500
400
300
200
100
0
561 r
474 r
501 r
483 s
488 s
Specific GR activity (U/ mg
protein)
GR activity of the selected w heat lines after F.
gram inearum infection
600
500
400
300
200
100
0
561 r
474 r
501 r
483 s
488 s
Some protein families (examples) which were induced during
Fusarium infection :
Zhou et al 2005
• Proteins with an antioxidant function: superoxide dismutase,
dehydroascorbate reductase, and glutathione S-transferases
(GSTs)
• Expression of proteins with highest similarity to
dehydroascorbate reductase and TaGSTF5 (a glutathione Stransferase) differed following FHB infection in susceptible
and resistant cultivars.
Specific GST activity (U/ mg
protein)
GST activity of the selected w heat lines
50
40
30
20
10
0
561 r
474 r
501 r
483 s
488 s
Specific GST activity (U/ mg
protein)
GST activity of the selected w heat lines after F.
culm orum infection
50
40
30
20
10
0
561 r
474 r
501 r
483 s
488 s
Specific GST activity (U/ mg
protein)
GST activity of the selected w heat lines after F.
gram inearum infection
50
40
30
20
10
0
561 r
474 r
501 r
483 s
488 s
FUSARIUM CULMORUM
Parameter
POD
SOD
KAT
GR
GST
Total ROS
Total antioxidant
capacity
561
474
501
483
488
FUSARIUM GRAMINEARUM
Parameter
POD
SOD
KAT
GR
GST
Total ROS
Total antioxidant
capacity
561
474
501
483
488
CONTROL
Parameter
POD
SOD
KAT
GR
GST
Total ROS
Total antioxidant
capacity
561
474
501
483
488
Conclusions:
The Fusarium inoculation on the ear influenced the hole
antioxidant system in the flag leaves of wheat in dough stage!
There were differences between the resistant and the sensitive
cultivars in POD activity inductions.
BUT according our measurements differences were detecable in
control circumstances.
And the amount of ROS, antioxidant capacity and the GST
activity correlated with the resistance.
Thank you for your attention!