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Influence of Pulsed Electric Field to Leaf Lettuce Evaluated onChlorophyll Fluorescence Measurement Using

Pulsed-Amplitude-Modulated Fluorometer

T. Sonoda1, Y. Higashi2, Y. Yamada1, D. Wang2, T. Namihira2, and H. Akiyama2

1Graduate School of Science and Technology, Kumamoto University, Japan2Institute of Pulsed Power Science, Kumamoto University, Japan

Abstract—Due to expectations to apply pulsed power technologies for various fields, applications of a pulsed electric field (PEF)on biological cells have been actively studied. In addition, the technique of chlorophyll fluorescence has become ubiquitous to revealphotosynthetic mechanism or stress reactions of photosynthetic organisms in ecophysiology studies in recent years. In this paper,chlorophyll fluorescence measurement was employed to reveal PEF effects to plants. Leaf lettuce was chosen as the materials ofthis experimental study and the subjects were treated with relatively low intensity of PEF, 0.2 kV/cm and relatively high intensityof PEF, 1.0 kV/cm, with 400 ns and 500 pulse. Experimental results using pulsed-amplitude-modulated fluorometer (PAM) showedthat 0.2 kV/cm has increased photosynthetic electron transport rate (ETR), and photochemical quenching (qP) that are related toredox state of electron acceptor in photosystem II. On the other hands, 1.0 kV/cm has decreased non-photochemical quenching(NPQ) that are related to heat dissipation of light energy.

Keywords—Pulsed electric field (PEF), leaf lettuce, chlorophyll fluorescence, PAM

I. INTRODUCTION

Physical phenomena in biological cells caused by externalpulsed electromagnetic energy, known bioelectrics, have avariety of applications on biotechnologies. Recently it hasattracted a lot of attention and developed over interdisciplinaryfields, especially, medical science and agriculture. Due toexpectations to apply pulse power technologies for both fields,applications of a pulsed electric field (PEF) on biological cellsis actively studied. Reference [1], [2] show that pore formationor increased permeability on a cell membrane occurred byexceeding a critical value of membrane voltage, known aselectroporation, and there is a possibility to be used for elec-trochemotherapy and gene delivery into cells. And also, somehave reported [3]–[5] that apoptosis, process of programmedcell death, can be activated by ns-PEFs in various cancercell lines in vitro. In addition, in agricultural field, there aresome reports including increased oil yield from plants [6], [7],improved juice extraction yield of fruits and root vegetables[8]–[10]. Furthermore, interestingly, some have reported thatplants growth can be stimulated by PEF. W. Songnuan andP. Kirawanich reported [11] pulsed electric field treatmentshowed that the effects appear significant the second weekafter treatments with a maximum increase of 80% comparedto the control for leaf area. R. Bovelli and A. Bennici reportedthe germination rate of Nicotiana tabacum was increasedby applying pulsed electromagnetic field [12]. Also, somereported [13], [14] the germination rate of Gladiolus bulbousor Arabidopsis seeds were increased by applying electricalstimulation. Although those studies suggest that PEF has a

Corresponding author: Douyan Wange-mail address: [email protected]

possibility to enhance plants growth, however, there are fewreports of a relationship between PEF and photosynthesis.

On the other hands, the technique of chlorophyll fluores-cence has become ubiquitous to reveal photosynthesis mech-anism or stress reactions of photosynthetic organisms in eco-physiology studies in recent years. The principle of chlorophyllfluorescence analysis [15]–[18] is relatively straightforward.Light energy absorbed by chlorophyll molecules, which useits energy to synthesize carbohydrates from CO2 and water,in a leaf can undergo one of three fates: photosynthesis(photochemical reaction), heat dissipation and chlorophyll flu-orescence (Fig. 1). These three process occur in competition,such that any increase in the efficiency of one will result in adecrease in the yield of the other two. Hence, by measuring theyield of chlorophyll fluorescence, information about changesin the efficiency of photochemical reaction and heat dissipation

Fig. 1. Schematic representation of a plant photosystem embedded in athylakoid membrane that undergoes electron transport for photosynthesis(photochemical reaction), heat dissipation and chlorophyll fluorescence.

Sonoda et al. 81

can be gained [15]. In addition, electron transport process,that occurs in the thylakoid membranes of chloroplasts duringphotosynthesis, is important parameter to understand photo-synthesis. It helps establish a proton gradient that powers ATPproduction and also stores energy in the reduced coenzymeNADPH which provides the reducing power to the Calvincycle to produce sugar and other carbohydrates.

In this study, for a basic research of PEF effects to plants,some parameters were measured by chlorophyll fluorescencefrom the leaf lettuce. For the evaluation, photosynthetic elec-tron transport rate (ETR), photochemical quenching (qP, re-lated to the redox state of electron acceptor in photosystemII) and non-photochemical quenching (NPQ, related to heatdissipation of light energy) were measured and discussed.

II. METHODOLOGY

A. Plant material and growth conditions

Leaf lettuce (Lactuca sativa L.) (Frillice, Snow BrandSeed Co., Ltd., Japan) was chosen as the materials of thisexperimental study because it is a typical item cultivatedin plant factories [19]. These seeds were germinated andgrown on sponge-like foamed urethane cuboids (2.3 cm width,2.3 cm depth, 2.8 cm height) steeped in liquid fertilizer (No.1and No.2 mixture of OTSUKA HOUSE, Otsuka AgriTechnoCompany, Japan) that was composed of 10% N, 8% P2O5,27% K2O, 4% MgO, 0.1% MnO, 0.1% B2O3, 0.18% Fe,0.002% Cu, 0.006% Zn, 0.002% Mo, 11% N, and 23% CaOin a plastic tray (34 cm width, 14 cm depth, 6 cm height)in an incubator (M-230F, TAITEC Company, Ltd., Japan).Plants were established at 20C day/night temperatures witha light-dark cycle of 12 h and 12 h (light illumination of 70-90 µmol/m2s) for 24 days. A certain amount of liquid fertilizerwas added to the plastic trays maintain the level to surface ofthe urethane cuboids due to evaporation during incubation.

B. Chlorophyll fluorescence measurements

Pulsed-amplitude-modulated fluorometer has been success-fully applied worldwide by numerous scientists in order toinvestigate the photosynthetic functions of plants. As men-tioned, light energy absorbed by chlorophyll molecules canundergo one of three fates: photosynthesis (photochemicalreaction), heat dissipation and chlorophyll fluorescence andthe equipments can obtain them. In this study, chlorophyllfluorescence of the samples were measured using pulsed-amplitude-modulated portable chlorophyll fluorometer (PAM-2500, Heinz Walz GmbH, Germany) on a leaf of the nurserylettuce and used the saturation pulse method that photosyn-thetically active radiation (PAR) was set to be between 0and 407 µmol/m2s, that allows determination of the quantumyields of photosystem II (φPS II). Saturated flash intensity was8700 µmol/m2s. ETR are derived from φPS II according toETR = PAR×φPSII×0.84×0.5 [20]. The 0.84 correspondsto the fraction of incident photons absorbed by photosyntheticpigments. The 0.5 corresponds to photons absorbed by PS IIrelative to photons absorbed by photosynthetic pigments. Plus,qP and NPQ were calculated by PamWin3 software that wasconnected to PAM-2500 during the measurement according to

Fig. 2. Schematic diagram of PFN used to generate PEF.

Fig. 3. Schematic diagram of electrodes and treatment chamber.

qP =(Fm′ − F

)/(Fm′ − Fo

′) and NPQ = Fm/Fm′ − 1

[17], [18], [21], where F , Fm, Fm′ and Fo

′ indicate fluores-cence intensity at any time, maximal fluorescence intensity inthe dark-adapted tissue, maximal fluorescence intensity in anylight adapted state and minimal fluorescence intensity withall PS II reaction centers open in any light adapted state,respectively.

C. Pulsed electric filed treatments

Fig. 2 is a schematic diagram of the self-manufactured PEFgenerator used to generate the single square pulse voltage inthis experiment. The generator consisted of a DC high-voltagesource (Model-600F, H.V Regulated D.C Power Supply, PulseElectronic Engineering Co., Ltd.) and a pulse forming network(PFN). A triggered spark gap switch (SGS) was used asthe closing switch for the PFN. The PFN had a 13-stageLC ladder, which was composed of 5.3 nF capacitors and133 nH inductors. The characteristic impedance (L/C) andthe pulse width (NLC) of PNF, calculated from capacitance(C) and inductance (L) of the LC ladder and the number (N )of LC ladder stages, were approximately 5 Ω and 400 ns,respectively. Furthermore, charging voltage was altered byadjusting the distance of Spark Gap Switch in order to generate0.2 kV.cm and 1.0 kV/cm electrical intensities. In Fig. 2, a 5 Ωregister was placed in order to absorb the ringing caused byinductance and capacitance after the initial voltage pulse. Inthe PEF technology, an pulsed electric field is applied to thenursery lettuce, put in perforated acrylic plate, placed on theacrylic plate between two electrodes (d = 150 mm) (Fig. 3).The two plate electrodes, fabricated of aluminum in a 70 mmsquare with thickness of 5 mm, are placed on the acrylicplate and the plastic tray filled with liquid fertilizer set under

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Fig. 4. Output waveforms of applied voltage and current through the electrodes.

the acrylic plate. Fig. 4 shows output waveforms of appliedvoltage and current through the treatment reactor. Appliedvoltage and current were monitored using a high-voltage probe(P6015A, Tektronix, Inc., USA) and a current probe (Pear-son current monitor, Model 2877, Pearson Electronics, Inc.),reading through a digital oscilloscope (DPO3054, Tektronix,Inc., USA). Electric field intensity was calculated by dividingapplied pulsed voltage by electrode distance. Before PEFtreatments, some samples were adapted to light conditions(70-90 µmol/m2s) in the incubator for at least 3 h, light-acclimated samples, and also some samples were adaptedto dark conditions in the incubator for at least 3 h, dark-acclimated samples. Prior to PEF treatment, both samplesare dark-adapted for 30 min to measure Fm [15]. After theadaptation, each sample was moved from the incubator to thetreatment chamber and put in the hole on the acrylic plateone at a time. Following this procedure, uniform PEF wasapplied to leaves of the sample. Applied PEF intensities were0.0 (for non-treated control), 0.2 and 1.0 kV/cm. Pulse numberwas fixed at 500 pulse under each treatment condition, andthe pulse repetition rate was fixed at 1 pps. To reduce thevariability among the samples, three or four samples wereused for each PEF treatment. Immediately after PEF treatment,chlorophyll fluorescence of the treated sample was measuredby PAM-2500.

D. Statistical analysisThe mean number of each calculated chlorophyll fluores-

cence parameters of treated samples was compared with thatof the untreated samples by Student’s t-tests (one-tailed pairedsample), using Microsoft Office Excel 2007, where the level ofsignificance was set at 0.05. The error bar for each replicationwas determined from standard error (SE).

III. RESULTS

A. Effect of PEF treatment on chloroplastic electron trans-port rate (ETR) in light-acclimated and dark-acclimated nurs-ery lettuce

To reveal the influence of PEF on electron transport rate(ETR) in the photosynthetic systems of light-acclimated and

dark-acclimated plants, leaves of the nursery leaf lettuceswere exposed to pulsed electric field of two different pulsedelectric intensities (0.2 kV/cm or 1.0 kV/cm) with 400 ns and500 pulse. Fig. 5 shows the results of ETR on each conditionsafter PEF treatment. There was no significant changes inETR with PEF treatment in the light-acclimated samples.On the other hands, 0.2 kV/cm treatment had a significantincreasing effect on the ETR in the dark-acclimated sampleswhereas 1.0 kV/cm treatment had no significant effect onit. For the representation of the change rate of the ETR indark-acclimated samples, a normalized form was used (Fig.6). Obviously, increased change rate of ETR was obtainedby applying 0.2 kV/cm in all observation points of PARwhile there were no significant ETR differences between1.0 kV/cm treatment and control. The maximum increase inthe change rate was obtained at 218 µmol/m2s (+35%). Thesesamples were cultivated under 90 µmol/m2s. Thus, 0.2 kV/cmtreatment was able to increase ETR under a broad range oflight illumination including the light conditions used for thelettuce cultivation in this study.

B. Effect of PEF treatment on photochemical quench-ing (qP) and non-photochemical quenching (NPQ) in dark-acclimated nursery lettuce

To obtain a deeper insight into the influence of the PEFtreatment to dark-acclimated nursery lettuce, photochemicalquenching (qP) and non-photochemical quenching (NPQ)were calculated during the measurement. For the representa-tion of the change rate of both in dark-acclimated samples, anormalized form was used (Fig. 7). As one can see, 0.2 kV/cmtreatment had a significant increase in qP compared withthe control whereas 1.0 kV/cm had no significance. Themaximum increase in change rate of qP treated with 0.2 kV/cmwas obtained at 218 µmol/m2s (+28%). On the other hands,1.0 kV/cm treatment had a significant decrease in NPQ com-pared with the control while there was no significance between0.2 kV/cm treatment and the control. The maximum decreasedchange rate of NPQ was obtained at 218 µmol/m2s (-36%).

Sonoda et al. 83

Fig. 5. The results of ETR on each conditions, light-acclimated and dark-acclimated, after PEF treatment.

Fig. 6. The change rate of the ETR in dark-acclimated samples.

IV. DISCUSSION

The results showed that chloroplastic electron transfer rate(ETR) of dark-acclimated nursery leaf lettuce can be increasedby 0.2 kV/cm PEF application but not by 1.0 kV/cm. Toinvestigate the influence of two different PEF intensities inphotosynthetic systems, photochemical quenching (qP) andnon-photochemical quenching (NPQ) were measured. Fromthese results, it was found that 0.2 kV/cm PEF application todark-acclimated nursery lettuce was effective for increasingETR and qP, in addition, 1.0 kV/cm PEF application waseffective for decreasing NPQ (Fig. 8). These results suggestthe following three possibilities.

1) 0.2 kV/cm treatment might promote the electron transferthrough PSII on leaves of the nursery lettuce.

2) 0.2 kV/cm treatment might promote the oxidation ofplastoquinone pool during the photochemical reaction.

3) 1.0 kV/cm treatment might inhibit the heat dissipationof absorbed light energy.

Regarding the possibility i) and ii), 0.2 kV/cm treatmentmight promote the oxidation of reduced plastoquinone. Thephotosynthetic apparatus of plants consists of two types ofphotosynthetic reaction centers: PSII and PSI. Both photosys-

tems are connected in series, with electrons flowing from PSIItoward PSI through an intermediate electron acceptor pool,which comprises the so-called plastoquinone (PQ) pool. Theredox potential of the PQ pool is clamped by the relative ratesof electron release into and uptake from this pool. Withinthe PSII complex, electrons are extracted from water at thelumenal side of the thylakoid membrane and transferred tothe primary plastoquinone electron acceptor (QA) [22]. Here,the photochemical capacity at PSII, which can be quantifiedby photochemical quenching (qP), is largely defined by theredox-state of the primary stable acceptor, QA [23] In thedark, QA is in a highly oxidized state. Under illumination,QA carries a single negative charge and is in a highly reducedstate [24]. In this study, qP was increased by 0.2 kV/cmtreatment, suggesting that the oxidation of plastoquinone mightbe promoted. Furthermore, nicotinamide adenine dinucleotidephosphate (NADPH), which is one of the essential moleculesfor the Calvin cycle, might be actively produced because thetaken electrons are used for its generation. If these anticipa-tions are true, the production of sucrose in the Calvin cyclemay be increased and growth stimulation of leaf lettuce canbe expected.

Regarding the possibility iii), non-photochemical quench-ing (NPQ) is related to heat dissipation of light energy.NPQ plays an important role in modulating photosyntheticperformance [25]. One of the causes of heat dissipation isthe de-epoxidation of xanthophylls in xanthophyll cycle inthylakoid membrane. The xanthophyll cycle activity has beenrelated to alterations in NPQ [26]. And also, some carotenoidsare involved in the xanthophyll cycle, which are relevantto thermal energy dissipation [27]. This cycle consists ofinterconversions between three carotenoids in the thylakoidmembrane: violaxanthin (V), antheraxanthin (A), and zeax-anthin (Z), and is ubiquitous in all land plants and greenalgae. Violaxanthin de-epoxidase catalyzes the light-inducedde-epoxidation of V to Z via A, and zeaxanthin epoxidasecatalyzes the epoxidation of Z to V via A on the dark [28].The de-epoxidation of xanthophyll is induced by acidificationof thylakoid lumen. Z. H. Zhang et al. [29] reported that PEF

84 International Journal of Plasma Environmental Science & Technology, Vol.11, No.1, APRIL 2017

Fig. 7. The change rate of photochemical quenching (qP) and non-photochemical quenching (NPQ) in dark-acclimated samples.

Fig. 8. A brief summary of the results.

treatment was increased the pH value of amino acid solutiondepending on the increase in both of the PEF intensity andpulse number. Furthermore, C. Cortes et al. [30] reported thatthe high-intensity pulsed electric fields (HIPEF) has effect onthe concentrations of total carotenoids in orange juice. It ispossible that the decreased NPQ in the nursery lettuce treatedwith 1.0 kV/cm PEF might be caused by the pH change ofthylakoid lumen or disruption of some carotenoids related tothe xanthophyll cycle.

V. CONCLUSION

For a basic research of PEF effects to plants, photosyntheticelectron transport rate (ETR), photochemical quenching (qP)and non-photochemical quenching (NPQ) are measured bychlorophyll fluorescence from leaves of leaf lettuce in thisstudy. Experimental results show that ETR and qP of dark-acclimated nursery leaf lettuce can be increased by 0.2 kV/cmPEF application while 1.0 kV/cm treatment had no signif-icant effect. On the other hands, NPQ can be decreasedby 1.0 kV/cm treatment while 0.2 kV/cm treatment had nosignificant effect. These results suggests that relatively lowPEF may improve the electron transfer flux through PSII inphotosynthesis, however, relatively high PEF may inhibit theheat dissipation which serve as stress defense. Interestingly,increased ETR or qP were observed under various light illumi-nation conditions including that used for the lettuce cultivation

in this study. It has a possibility that leaf lettuce can be grownunder relatively low illumination using the PEF treatment.Detailed investigation of PEF influence to photosynthesis inplants is required as future work.

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