Antioxidant activities and reactive oxygen species in

flag leaves of Fusarium-inoculated wheat genotypes Szeged – Timişoara axis for the safe food and feed

SZETISA1HURO/0901/147/2.2.2

Ágnes GalléDepartment of Plant Biology

University of Szeged

2nd Progress Report MeetingSzeged, 22-23 September, 2011

Reporting period: 01.06. 2011 – 30.09. 2011

www.huro-cbc.euwww.hungary-romania-cbc.eu

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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.

Before inoculation sampling (ROS and total antioxidant capacity)

anthesis

inoculation

SAMPLING

Dough stagePremilk stage Milk stage

GK Mini Manó/Nobeokabozu genetic mapping population (Prof. Mesterházy Ákos )

Nobeokabozu: FHB resistant lineA recombinant line from China: ‘WSY’, Wangshubai/Sumai3/Yangangfanzhou

GK Mini ManóArthur 71 / Sava // Rusalka / NS 171.2 /3/ F 30.74Resistant line to e.g. leaf rust

Plant material

Total intracellular ROS before inoculation

0

100

200

300

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800

900

474 r 501 r 483 s 488 s

DC

F fl

uo

resc

ence

Total intracellular ROS after inoculation with F.c.

0

100

200

300

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800

900

561 r 474 r 501 r 483 s 488 s

DFC

fluor

esce

nce

Total intracellular ROS after inoculation with F.g.

0

100

200

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900

561 r 474 r 501 r 483 s 488 s

DFC

fluor

esce

nce

Total intracellular ROS after inoculation

0

100

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900

561 r 474 r 501 r 483 s 488 s

DCF

fluor

esce

nce

Antioxidant activity (FRAP)

0

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30004000

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7000

8000

561 r 474 r 501 r 483 s 488 s

Ant

ioxi

dant

act

ivity

(m

mol

FeS

O4/

g FW

)

Antioxidant activity (FRAP)After inoculation with F. c.

0

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30004000

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7000

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561 r 474 r 501 r 483 s 488 sA

ntio

xida

nt a

ctiv

ity

(mm

ol F

eSO

4/ g

FW)

Antioxidant activity (FRAP)After inoculation with F. g.

0

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561 r 474 r 501 r 483 s 488 s

Ant

ioxi

dant

act

ivity

(m

mol

FeS

O4/

g FW

)

Antioxidant activity (FRAP)Before inoculation

0

1000

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6000

474 r 501 r 483 s 488 s

Ant

ioxi

dant

act

ivity

(m

mol

FeS

O 4/ g

FW

)

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)

POD activity of the selected wheat lines

0

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561 r 474 r 501 r 483 s 488 sSpec

ific

POD

act

ivity

(U/ m

g pr

otei

n)

POD activity of the selected wheat lines after F. culmorum infection

0500

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20002500

3000

561 r 474 r 501 r 483 s 488 sSpec

ific

POD

act

ivity

(U/ m

g pr

otei

n)

POD activity of the selected wheat lines after F. graminearum infection

0

5001000

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561 r 474 r 501 r 483 s 488 sSpec

ific

POD

act

ivity

(U/ m

g pr

otei

n)

SOD activity of the selected wheat lines

0246

8101214

561 r 474 r 501 r 483 s 488 sSpec

ific

SOD

act

ivity

(U/ m

g pr

otei

n)

SOD activity of the selected wheat lines after F. culmorum infection

02468

101214

561 r 474 r 501 r 483 s 488 sSpec

ific

SOD

act

ivity

(U/ m

g pr

otei

n)

SOD activity of the selected wheat lines after F. graminearum infection

02468

101214

561 r 474 r 501 r 483 s 488 sSpec

ific

SOD

act

ivity

(U/ m

g pr

otei

n)

KAT activity of the selected wheat lines

0

100

200

300

400

500

600

561 r 474 r 501 r 483 s 488 sSpec

ific

KA

T ac

tivity

(U/ m

g pr

otei

n)

KAT activity of the selected wheat lines after F. culmorum infection

0

100

200300

400

500

600

561 r 474 r 501 r 483 s 488 sSpec

ific

KA

T ac

tivity

(U/ m

g pr

otei

n)

KAT activity of the selected wheat lines after F. graminearum infection

0

100

200

300

400

500

600

561 r 474 r 501 r 483 s 488 sSpec

ific

KA

T ac

tivity

(U/ m

g pr

otei

n)

GR activity of the selected wheat lines

0

100

200

300

400

500

600

561 r 474 r 501 r 483 s 488 s

Spec

ific

GR

act

ivity

(U/ m

g pr

otei

n)

GR activity of the selected wheat lines after F. culmorum infection

0

100

200

300

400

500

600

561 r 474 r 501 r 483 s 488 sSpec

ific

GR

act

ivity

(U/ m

g pr

otei

n)

GR activity of the selected wheat lines after F. graminearum infection

0

100

200

300

400

500

600

561 r 474 r 501 r 483 s 488 sSpec

ific

GR

act

ivity

(U/ m

g pr

otei

n)

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 S-transferase) differed following FHB infection in susceptible and resistant cultivars.

GST activity of the selected wheat lines

0

10

20

30

40

50

561 r 474 r 501 r 483 s 488 sSpec

ific

GST

act

ivity

(U/ m

g pr

otei

n)

GST activity of the selected wheat lines after F. culmorum infection

0

10

20

30

40

50

561 r 474 r 501 r 483 s 488 sSpec

ific

GST

act

ivity

(U/ m

g pr

otei

n)

GST activity of the selected wheat lines after F. graminearum infection

0

10

20

30

40

50

561 r 474 r 501 r 483 s 488 sSpec

ific

GST

act

ivity

(U/ m

g pr

otei

n)

Parameter 561 474 501 483 488

POD

SOD

KAT

GR

GST

Total ROS

Total antioxidant capacity

FUSARIUM CULMORUM

Parameter 561 474 501 483 488

POD

SOD

KAT

GR

GST

Total ROS

Total antioxidant capacity

FUSARIUM GRAMINEARUM

Parameter 561 474 501 483 488

POD

SOD

KAT

GR

GST

Total ROS

Total antioxidant capacity

CONTROL

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!