Antioxidative Activity of Plant and

Regulation of Plant Growth Induced by

Oxygen Radicals

As traditional legend, the lighting strike enhances the growth the rice plant. This character means “partner of rice plant”. We have used the electrical stimulus for agriculture from ancient.

Stimulation to plantsStimulation to plants

Current stimulation method for plant growth is using chemical agents, that is, plant hormone.

Germination and growth enhancement of plants have been performedby chemical agents with the function of plant hormone such as gibberellin.

Major plant growth regulate agents are below:

Enhancement of germination and rooting

Indolebutyric acid

1-Naphthylacetamide

Growth inhibitor

Prohexadione-Calcium

Chlormequat

Promotion of flowering and fruition

Gibberellin

Cloxyfonac

There are possibility of risks of residual effect (persistence).

Current stimulation method for plant growthCurrent stimulation method for plant growth

Above figure is radish sprouts with irradiation of atmospheric oxygen plasma after 4 days of cultivation. This result implies unknown reaction of a living body.

plasma irradiationplasma irradiationcontrolcontrol

Detailed phenomena appeared on living bodyand the mechanism are still unknown.

Physical impactChemical reaction

Purpose

Purpose:Characteristics and mechanism of growth enhancement of plantsby plasma irradiation are attempted to be clarified from theviewpoint of antioxidative characteristics of living body.

The effect of growth enhancement is obtained by the plasma irradiation to seeds of plants. After seedling, there are not any stimulations to the plants.

Sample Gas : O2Pressure : 60 PaInput RF power : 60 WTreatment time : 30 120 min

RF plasma device and OES at the position of seeds varying O2 pressure

seeds on dish

450 [mm]

RFPowersupply8

[inch

]

OO22

CCP antenna

VacuumpumpStainless

Chamber

400 500 600 700 800 900 10000

2000

4000

6000

8000

10000 O 2 gas pressure 20 Pa 40 Pa 60 Pa 80 Pa

Inte

nsir

y (a

rb.u

nits

)W avelength (nm)

The relation between the emission intensity at wavelengths of 761nm and 777nm and oxygen pressure.

10 20 30 40 50 60 70 80 900

2000

4000

6000

8000

10000 1g+ at 761nm

O(3P) at 777nm

Inte

nsity

(arb

.uni

ts)

Pressure (Pa)

At 60 Pa, dominant particle is excited oxygen molecule 1

g+.

Variation of exterior of seeds after plasma irradiationVariation of exterior of seeds after plasma irradiation

without plasma irradiation plasma irradiation for 30min

When the seed is immersed in water, both the treated and untreated husk get wet in a few minutes. So, the hydrophilicity of husk does not relate to the germination enhancement.

There is no significant damage on the husk of seed.

Response to the plasma stimulus and reactions induced by plasma would occur inside the seed husk.

Effect on sprout germinationEffect on sprout germination

Atmospheric plasmaPower consumption 60 W / Gas OxygenIrradiation period 30 min / Temp. 21.5 oCSample : Raphanus sativus var. longipinnatus 'Kaiwaredaikon'

24 h after seeding

O2 plasma irradiation to seedscontrol

Effect on germination of sproutEffect on germination of sprout

1 2 30

20

40

60

80

100

Ger

min

atio

n ra

te (%

)

Cultivation period (day)

plasma irradiation control

Germination rates of Raphanus sativussprout with and without plasma irradiation.

Final germination ratecontrol (untreated) 90%irradiation for 30 min 95%irradiation for 60 min 95%

Final germination rate was not affected by the plasma irradiation.

In case of control, germination rate is 30% in one day after the seeding. In case of plasma irradiation, the rate increases to 80–90%. Plasma irradiation induces the germination, which leads to uniform growth of plants.

Growth enhancement of radish sproutGrowth enhancement of radish sprout

without plasma irradiation

plasma irradiation for 30min

Root length increased

Chlorophyll increased

48 h after seeding Atmospheric plasmaPower consumption 60 W / Gas OxygenIrradiation period 30 min / Temp. 21.5 oCSample : Raphanus sativus var. longipinnatus 'Kaiwaredaikon'

Temporal variation of sprout length after plasma irradiationTemporal variation of sprout length after plasma irradiation

0 30 600

20

40

60

80

100

120

140

160

180

99%

131%

156%

10

Ave

rage

d sp

rout

leng

th (%

)

Plasma irradiation period (min)

96 h after seeding

Sprout length becomes 1.5 times ofthe original length in 4 days after theirradiation.

0 20 40 60 80 1000

1

2

3

4

5

6

7

8

9 Air plasmaO2 plasma

Ave

rage

spro

ut le

nght

(cm

)

Cultivation period (h)

Material gas Oxygen, Air

Oxygen radicals is the importantfactor for the enhancement effect.

Reactions of intracellular substances of plantsReactions of intracellular substances of plantsinduced by plasma irradiationinduced by plasma irradiation

Reactions of intracellular substances are investigated.

Removal of intracellular active oxygenRemoval of intracellular active oxygen

Plant cell

AntioxidantsAntioxidantsgenerationgeneration

Active species Active species from plasmafrom plasma

Removal of Removal of excessexcessoxygenoxygen

Association of glutathione with flowering in Arabidopsis thalianaOgawa K, Tasaka Y, Mino M, Tanaka Y, Iwabuchi M., Plant Cell Physiol. 2001 May;42(5):524-30.

Removal of excess active oxygens by plasma irradiation leads togrowth enhancement of plants.

Excess active oxygens generates lipid peroxides

Antioxidant activity varying the plasma Antioxidant activity varying the plasma irradiation period.irradiation period.

Antioxidative activity of seed induced by oxygen plasmaAntioxidative activity of seed induced by oxygen plasma

0 5 10 15 20 25 300.0

0.5

1.0

1.5

2.0

Ant

ioxi

dant

act

ivity

(arb

.uni

ts)

Plasma irradiation period (min)

DPPHmethod

Quantification of antioxidative activity Quantification of antioxidative activity -- DPPH methodDPPH method

Oxygen plasma irradiation increases antioxidative activity in seed.

Active oxygen sensor: redox reactions of thiol compoundsActive oxygen sensor: redox reactions of thiol compounds

※

Structure

HOOCO

NH

SH

O

HN COOH

NH2 reduction type

HOOCO

NH

S

O

HN COOH

NH2

COOHO

HN

S

O

NH

HOOC

NH2

oxidation typeoxidation type

Thioredoxin Gultathione

Whole picture from plasma irradiation to growth enhancementWhole picture from plasma irradiation to growth enhancement

Thiol compoundsThiol compounds

Active oxygen gene expression

dissociationdissociationregulation

Redox reaction and activation of transcription factor for plantRedox reaction and activation of transcription factor for plant growthgrowth

Sensor of activeSensor of activeoxygensoxygens

““ThioredoxinThioredoxin””

SH

TrXTrX

SH S S

TrXTrXTranscription factorTranscription factorfor oxidative stressfor oxidative stress

production andactivation of MAPK

Transcription factorTranscription factorfor oxidative stressfor oxidative stress

activation oftranscription factor

antioxidativeantioxidativesubstancessubstances

cell cyclecell cycleaccelerationacceleration

0 5 10 15 20 25 300.0

0.5

1.0

1.5

2.0

Thio

l am

ount

(arb

.uni

ts)

Treatment period (min)

Air Plasma

0 10 20 30 40 50 600.0

0.5

1.0

1.5

2.0

Thio

l am

ount

(arb

.uni

ts)

Treatment time (min)

N2 Plasma

Relation between thiol quantity and growth enhancementRelation between thiol quantity and growth enhancement

0 5 10 15 20 25 30 350

2

4

6

8

10

Plasma irradiation period (min)

Ave

rage

d sp

rout

leng

th (c

m)

Averaged length of sprout0

2

4

6

8

10

Thiol amount (arb. units)

Thiol quantity

-5 0 5 10 15 20 25 30 350

2

4

6

8

10

114%

151%159%

Ave

rage

spro

ut le

ngth

(cm

)

Plasma irradiation period (min)

Relation between thiol quantity and growth enhancementRelation between thiol quantity and growth enhancement

Active oxygen irradiation(oxidative stress)

Oxidation of Thiol compounds

Activation of transcription factor

Activation of transcription factor andbind to DNA

Enzyme production

Plant hormone production

Growth enhancement of plantsGrowth enhancement of plants

Removal of active oxygen

Production of antioxidative substances

-SH -S-S-

Speculative route from plasma irradiation to growth enhancementSpeculative route from plasma irradiation to growth enhancement

Activation of Mitogen activated Protein Kinase

Blue parts are confirmedby experiments

Investigation of genetic mutation

It is important to investigate the genetic mutation, we use Arabidopsisof wild-type for scientific purpose and practical use of this technique.

Growth enhancement of Arabidopsis by plasma irradiationGrowth enhancement of Arabidopsis by plasma irradiation

control plasmairradiation

50 h

230 h

0 50 100 150 200 2500

20

40

60

80

100

120

140

160

Nor

mal

ized

scap

e le

ngth

(%)

Incuvation period from irradiation (h)

Atmospheric plasma

Arabidopsis thaliana Columbia 01Arabidopsis seed

Arabidopsis thaliana – model plant

Control Plasmairradiation30min

Plasmairradiation3min

Arabidopsis thalianaColumbia 01

Effect of multiple irradiation to seeds growth enhancement

Irradiation twice

Irradiation once

Untreated(wild-type)

Variation of leaf size after the multipleirradiation of plasma.

untreated Irradiation twice

twice once untreated

Multiple irradiation growth enhancement

Sample Gas : O2Pressure : 60 PaInput RF power : 60 WTreatment time : 30 120 min

Arabidopsiswild type(Columbia 01)

Dependence of total length of plant on the number of times of plasma irradiation.

Multiple irradiation length of leaf and scape

0 1 20

50

100

150

200 length of leaf length of scape

Leng

th (m

m)

Number of times of irradiationuntreated

3

Both leaf and scape length increases with the number of times of the plasma irradiation.

This fact implies that the genetic mutation by plasma irradiation occurs, and information of the growth enhancement is reserved in DNA.

Irradiation of excimer UV light to seeds

untreated

Ozone +excimer UV (172nm)

Auxin-like effect

Variation of root growth

Variation of root growth

Sterilization of Agricultural Products

Using Oxygen Radicals Produced by

Barrier Discharge

The advantage of plasma sterilizationToxicity freePersistence freeNo generation of resistant bacteria

0.5mm

4mm

37mm

15mm

6mm

15mm

Surface discharge

UV

Vis

Surface discharge and UV light

Sample setting

Production of active oxygen using discharge and UV light

+

+

Ozone concentration and generation of oxidants

Ozoneconcentration CI (5min) CI (10min) CI (20min)

Spore of Aspergillus Oryzae

w/o plasma irradiation 30 min plasma irradiation

w/o plasma irradiation 30 min plasma irradiation

Swab test and colony production

Spore of yeast / mold

Ozone 60ppm UV 254nm UV + ozoneControl

0 5 10 15 20

20

40

60

80

100

Inac

tivat

ion

rate

(%)

Plasma irradiation period (min)

plasma irradiationfor 5 minw/o treatment plasma irradiation

for 10minplasma irradiation

for 20min

Sterilization of lemon surface using discharge and UV light

1000 2000 3000 4000

0.00

0.05

0.10

0.15

0.20

without irradiationafter irradiation

Abs

orba

nce

(arb

.uni

ts)

Wavenumber (cm-1)

ATR FTIR spectra of Lemon peel surfaceAtmospheric torch plasma+UV light irradiation

N-H stretchingC-Cstretching C=O stretching

Amide I

C-H stretching

w/o plasma

plasma irradiation

Plasma irradiation to seeds of rice plantOxygen RF plasma, 60Pa, 60W, 30 - 90min

Plasma sterilizer

Rice seeds

0 20 40 60 80 1000

20

40

60

80

100

Inac

tivat

ion

rate

of m

old

Plasma irradiation period (min)

moldE. coil

E. coli

Inactivation rate of mold and E. coli varying treatment period.Oxygen RF plasma, 60Pa, 60W, 30 - 90min

Control

30min

90min

Mold on culture sheet.

ricerice

Control 60min

120min 180min

Sterilization of seed surface

0 20 40 60 80 100 120 140 160 180 2000

20

40

60

80

100

carrotcarrot

material gas oxygen plasma air plasma Ar plasma

Inac

tivat

ion

rate

of m

old

Plasma irradiation period (min)

Inactivation rate of mold varying treatment period. Oxygen RF plasma, 60Pa, 60W, 60 - 180min

rapidsterilization

Inactivation of Plant virus and RNA

Withouttreatment

Atmospheric plasmairradiation

Plasma irradiation to water with Cucumber mosaic virus RNA.

Power input 60 WGas : oxygenGas flow rate : 1.0 L/minTreatment period : 20 min

Withouttreatment

Atmospheric plasmairradiation

Inactivation of virus RNA

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• B1 B2365nm 450nm

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Future worksFuture works