Antioxidative Activity of Plant and Regulation of Plant Growth Induced by Oxygen Radicals

Transcription

1 AntioxidativeActivityofPlantand RegulationofPlantGrowthInducedby OxygenRadicals Stimulation to plants 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. Current stimulation method for plant growth is using chemical agents, that is, plant hormone.

2 Current stimulation method for plant growth Germination and growth enhancement of plants have been performed by 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). Purpose control plasma irradiation Physical impact Chemical reaction Detailed phenomena appeared on living body andthemechanismarestillunknown. Purpose: Characteristics and mechanism ofgrowthenhancementofplants by plasma irradiation are attempted to be clarified from the viewpointofantioxidativecharacteristics oflivingbody. Above figure isradish sprouts with irradiation of atmospheric oxygen plasma after 4 days of cultivation. This result implies unknown reaction of a living body.

3 RF plasma device and OES at the position of seeds varying O2 pressure 45 [mm] O 2 Intensiry (arb.units) O 2 gas pressure 2 Pa 4 Pa 6 Pa 8 Pa 8 [inch] CCP antenna RF Power supply Wavelength (nm) Stainless Chamber Vacuum pump Sample Gas : O 2 Pressure : 6 Pa Input RF power : 6 W Treatment time : 3 12 min seeds on dish 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. Intensity (arb.units) 1 1 g + at 761nm O( 3 P) at 777nm Pressure (Pa) The relation between the emission intensity at wavelengths of 761nm and 777nm and oxygen pressure. At 6 Pa, dominant particle is excited oxygen molecule 1 g+. Variation of exterior of seeds after plasma irradiation 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. 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. without plasma irradiation plasma irradiation for 3min

4 Effect on sprout germination 24 h after seeding Atmospheric plasma Power consumption 6 W / Gas Oxygen Irradiation period 3 min / Temp o C Sample : Raphanus sativus var. longipinnatus 'Kaiwaredaikon' control O 2 plasma irradiation to seeds Effect on germination of sprout 1 Germination rate (%) plasma irradiation control Cultivation period (day) Germination rates of Raphanus sativus sprout with and without plasma irradiation. Final germination rate control (untreated) 9% irradiation for 3 min 95% irradiation for 6 min 95% Final germination rate was not affected by the plasma irradiation. In case of control, germination rate is 3% in one day after the seeding. In case of plasma irradiation, the rate increases to 8 9%. Plasma irradiation induces the germination, which leads to uniform growth of plants.

5 Growth enhancement of radish sprout 48 h after seeding Atmospheric plasma Power consumption 6 W / Gas Oxygen Irradiation period 3 min / Temp o C Sample : Raphanus sativus var. longipinnatus 'Kaiwaredaikon' without plasma irradiation plasma irradiation for 3min Chlorophyll increased Root length increased Temporal variation of sprout length after plasma irradiation Averaged sprout length (%) h after seeding 131% 99% 156% Average sprout lenght (cm) Material gas Oxygen, Air Air plasma O 2 plasma Plasma irradiation period (min) Cultivation period (h) Sprout length becomes 1.5 times of the original length in 4 days after the irradiation. Oxygen radicals is the important factorfortheenhancementeffect.

6 Reactions of intracellular substances of plants induced by plasma irradiation Reactions of intracellular substances are investigated. Removalofintracellularactiveoxygen Excess active oxygens generates lipid peroxides Removalofexcessactiveoxygensbyplasmairradiationleadsto growthenhancementofplants. Association of glutathione with flowering in Arabidopsis thaliana Ogawa K, Tasaka Y, Mino M, Tanaka Y, Iwabuchi M., Plant Cell Physiol. 21 May;42(5): Active species from plasma Antioxidants generation Plant cell Removal of excess oxygen

7 Antioxidative activity of seed induced by oxygen plasma Quantification of antioxidative activity - DPPH method DPPHmethod Antioxidant activity (arb.units) Plasma irradiation period (min) Antioxidant activity varying the plasma irradiation period. Oxygen plasma irradiation increases antioxidative activity in seed. Active oxygen sensor: redox reactions of thiol compounds Thioredoxin Gultathione HOOC O SH H N Structure COOH NH 2 N H O reductiontype HOOC HOOC NH 2 N H O O S S N H H N O O H N NH 2 COOH COOH oxidationtype

8 Whole picture from plasma irradiation to growth enhancement Redoxreactionandactivationoftranscriptionfactorforplantgrowth SH TrX SH regulation Transcriptionfactor foroxidativestress S S TrX dissociation Transcriptionfactor foroxidativestress productionand activationofmapk Sensorofactive oxygens Thioredoxin Active oxygen activationof transcriptionfactor geneexpression Thiolcompounds antioxidative substances cellcycle acceleration Relation between thiol quantity and growth enhancement 2. N 2 Plasma 2. Air Plasma Thiol amount (arb.units) Thiol amount (arb.units) Treatment time (min) Treatment period (min)

9 1 Averaged sprout length (cm) Relation between thiol quantity and growth enhancement 1 Average sprout length (cm) % % Plasma irradiation period (min) 114% Thiol quantity Averaged length of sprout Plasma irradiation period (min) Thiol amount (arb. units) Speculative route from plasma irradiation to growth enhancement Activeoxygenirradiation (oxidativestress) -SH -S-S- OxidationofThiolcompounds Blue parts are confirmed by experiments Activationoftranscriptionfactor ActivationofMitogenactivatedProteinKinase Activationoftranscriptionfactorand bindtodna Enzymeproduction Productionofantioxidativesubstances Planthormoneproduction Removalofactiveoxygen Growthenhancementofplants

10 Investigation of genetic mutation It is important to investigate the genetic mutation, we use Arabidopsis of wild-type for scientific purpose and practical use of this technique. GrowthenhancementofArabidopsisbyplasmairradiation Arabidopsisthaliana modelplant Arabidopsisseed Atmosphericplasma ArabidopsisthalianaColumbia1 5h 16 Arabidopsisthaliana Columbia1 Normalized scape length (%) h Incuvation period from irradiation (h) Control Plasma irradiation 3min Plasma irradiation 3min control plasma irradiation

11 Effect of multiple irradiation to seeds growth enhancement Untreated (wild-type) Irradiation once Irradiation twice Multiple irradiation growth enhancement Sample Gas : O 2 Pressure : 6 Pa Input RF power : 6 W Treatment time : 3 12 min untreated Irradiation twice Arabidopsis wildtype (Columbia1) twice once untreated Variation of leaf size after the multiple irradiationofplasma.

12 Multiple irradiation length of leaf and scape 2 length of leaf length of scape Length (mm) untreated Number of times of irradiation Dependence of total length of plant on the number of times of plasma irradiation. 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

13 Variation of root growth untreated Ozone +excimer UV (172nm) Auxin-like effect

14 Variation of root growth SterilizationofAgriculturalProducts UsingOxygenRadicalsProducedby BarrierDischarge

15 Theadvantageofplasmasterilization Toxicityfree Persistencefree Nogenerationofresistantbacteria Surface discharge 37mm 4mm 15mm.5mm 6mm 15mm

16 Surface discharge and UV light Vis Sample setting UV Production of active oxygen using discharge and UV light + +

17 Ozoneconcentrationandgenerationofoxidants Ozone concentration CI (5min) CI (1min) CI (2min) w/oplasmairradiation 3minplasmairradiation Spore of Aspergillus Oryzae

18 w/oplasmairradiation 3minplasmairradiation Swab test and colony production Sporeofyeast/mold Control Ozone6ppm UV254nm UV+ozone

19 Sterilization of lemon surface using discharge and UV light w/otreatment plasmairradiation for5min plasmairradiation for1min plasmairradiation for2min 1 Inactivation rate (%) Plasma irradiation period (min).2 without irradiation after irradiation Absorbance (arb.units) C-C stretching C=O stretching Amide I C-H stretching N-H stretching w/o plasma Wavenumber (cm -1 ) plasma irradiation ATR FTIR spectra of Lemon peel surface Atmospheric torch plasma+uv light irradiation

20 Plasma sterilizer Plasma irradiation to seeds of rice plant Oxygen RF plasma, 6Pa, 6W, 3-9min Rice seeds E.coli Control rice 1 3min 9min Inactivation rate of mold Plasma irradiation period (min) mold E. coil Inactivation rate of mold and E. coli varying treatment period. Oxygen RF plasma, 6Pa, 6W, 3-9min Mold on culture sheet.

21 Sterilization of seed surface carrot rapid sterilization 1 Control 12min 6min 18min Inactivation rate of mold material gas 2 oxygen plasma air plasma Ar plasma Plasma irradiation period (min) Inactivation rate of mold varying treatment period. Oxygen RF plasma, 6Pa, 6W, 6-18min Inactivation of Plant virus and RNA

22 Inactivation of virus RNA Plasma irradiation to water with Cucumber mosaic virus RNA. Power input 6 W Gas : oxygen Gas flow rate : 1. L/min Treatment period : 2 min Without treatment Atmospheric plasma irradiation Without treatment Atmospheric plasma irradiation Future works B1B2 365nm45nm