Jpn. J. Appl. Phys. Vol. 39 (2000) pp. 3696–3700Part 1, No. 6A, June 2000c©2000 The Japan Society of Applied Physics

Early Detection of Salt Stress Damage by Biophotons in Red Bean SeedlingTomoyuki OHYA, Hideaki KURASHIGE, Hirotaka OKABE and Shoichi KAI

Department of Applied Physics, Faculty of Engineering, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

(Received November 5, 1999; accepted for publication March 7, 2000)

The optical detection of the stress damage to plants by NaCl solutions was attempted during germination of a seed andgrowth of a root. We compared the photon intensity of red beans before and after NaCl treatment and found that the photonintensity after NaCl treatment decreased as the NaCl concentration increased. For the saturated NaCl concentration (4.5 M),however, the observed photon intensity drastically increased, and the simultaneous destruction of cell membranes was observed.The intensity of biophoton emission from red beans showed characteristic change with salt concentrations. When the salt stresswas applied to the red beans at an early growth stage, their root elongations were suppressed and photon intensity from the rootdecreased. This was not the case for the root at the late stage. This shows that biophoton intensity due to salt stress depends onnot only NaCl concentration but also the growth stage of the plant. We may conclude that the extent of damage to roots by saltstress can be evaluated from biophoton response.

KEYWORDS: biophoton, optical detection, salt stress, growth stage

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1. Introduction

It has been revealed that various environmental stressessuch as salt, drought, cold and osmotic stresses caused phys-iological changes in plants due to an increase in abscisic acid(ABA) concentration and the syntheses of proteins and en-zymes.1–4) Molecular biology has been a powerful tool for thestudy of stress responses of plants. However, the relationshipbetween physiological changes and microscopic response, forexample, what kinds of biochemical reactions were inducedby proteins and enzymes and how they succeed a series ofstress reactions, has not been well understood.

To understand the relation, it is necessary to obtain infor-mation concerning the biochemical reactions in intact plants.Therefore, we expect to detect without any injury physio-logical changes caused by some environmental stresses. Inorder to realize a nondestructive and noncontact measure-ment, biophoton measuring methods are preferable for detect-ing physiological informationin situ.5,6) Biophoton emissionis an ultraweak photon emission from living organisms, andis generated from excited molecular series such as reactiveoxygen series (ROS) which result from chemical reactionsin order to maintain homeostasis.7,8) In addition, endogenousROS are synthesized not only in metabolic processes but alsoin self-defensive reactions such as hypersensitive ones.9,10)

When plants are attacked by viruses, moreover, the endoge-nous ROS level immediately increases and viruses are sealedin cells necrotized by ROS.11) In such a process strong bio-photon radiation has been observed.12) On the other hand,since ROS also bring serious oxidative damage to plant cellsthemselves, plants have ROS scavenger systems such as su-peroxide dismutase (SOD) or catalase to protect them fromROS.10,13,14)

Very recently, we demonstrated that the environmental re-sponse of plants can be inferred from biophoton emission.15)

We focus on salt stress as the environmental stress in thepresent study, because it has caused serious damage to agri-cultural products. The detailed mechanism of growth inhibi-tion is still unclear. The growth dynamics of the germinationprocess are easy to model theoretically because of the sim-plicity of comparing with processes in adult plants. In orderto test how salt stress inhibits growth and how we could detectit from biophoton information, the process of germination of

two-dimensional photon counting system (Hamamatsu Pho-tonics PIAS-TI500 and C-1809) to detect local photon radia-tion and its spatial distribution.

2.2 Microscopic observation of cellsA root apex was cut to a layer thickness of 3 cells by a ra-

zor, and mounted on the slide glass. The root cells in the mid-dle layer were observed under an optical microscope (NikonLABOPHOT). Then NaCl solution was dropped on the sam-ple, and all processes were recorded onto a videotape.

3. Results and Discussion

3.1 Dependence of growth on NaCl concentrationThe seeds were cultivated in the following manner. Pure

water was used initially for 72 h as a cultivated solution, andit was replaced with NaCl solutions of various concentrations(Fig. 1). In pure water, the evolution curve of root length (thereference data) is well described by the logistic equation, asreported previously.16) In 0.01 M-NaCl solution, the growthcurve is not affected and has basically the same aspect as thereference data. On the other hand, in 0.1 M-NaCl solution, theelongation of roots was immediately stopped and root cellswere seriously damaged. These results clearly suggest thatthe salt stress of 0.1 M causes physiological damage and in-

red beans is a good candidate since red bean growth dynamicshave been well studied.16)

2. Experimental

The sample red bean seeds (Vigna angularis) were chosento be within the standard deviation of their weight distribution(the total number of seeds was 3,000) to exclude exceptionalones. These beans were placed for 24 h under conditions ofhumidity H = 95% and temperatureT = 35◦C to triggergermination, and then set in a growth chamber withH =82%, T = 24◦C at timet = 0. The whole culture of seedswas carried out in a darkroom to avoid photosynthesis.

2.1 Biophoton measurementThe details of the photon measurements have been de-

scribed elsewhere.16,18) Therefore, we will describe themvery briefly here. We used a universal photon counting sys-tem (Hamamatsu Photonics C-2550, photomultiplier R649)to measure the temporal change of biophoton emission, and a

hibits the growth of roots. In 1 M-NaCl solution, the rootelongation was immediately stopped. Moreover, root growthdid not recover in pure water upon removing the stress af-ter 1 M salt stress for 60 h. Namely,tela necrosisoccurred.Therefore, we named this concentration the lethal stress con-centration. Table I shows the summary of the results.

In order to investigate basic photon response with vari-ous NaCl concentrations, we measured photon radiation froma red bean att = 168 h when the root elongation processslowed, i.e., at the steady state of root growth. Photon mea-surements were performed for 60 h to evaluate whether plantscould acclimatize to the new environment or not. The tempo-ral changes of biophoton intensity for various concentrationsof NaCl (0 M, 0.01 M, 0.1 M, 1 M, 4.5 M (saturated concen-tration)) are shown in Fig. 2. The vertical axis shows a nor-malized excess photon intensityI which is defined by eq. (1).

I = (X/X0 − 1)× 100 (1)

Here, X0 is photon counts per hour averaged over for 5.5 hjust before the application of NaCl stress, andX is photoncounts per hour for 4 h with NaCl solutions.

There was no significant difference between the results for0 M and 0.01 M. It seems that roots fortg = 168 h were littleaffected at this concentration, that is, they could acclimatizeto the environmental change. Photon intensity decreased inthe case of the NaCl treatment at concentrations of 0.1 M and

1 M. It drastically increased, however, in the case of 4.5 M-NaCl treatment. This suggests that physiological changes ofplants by salt stress occur in the concentration range from1 M to 4.5 M. In order to study the phenomenological as-pects, we made microscopic observations of plant cells at theroot. Plasmolysis occurred immediately upon application ofa drop of 4.5 M-NaCl solution, and cell membranes were de-stroyed after 6 min as a result of osmotic pressure and chem-ical decomposition (Fig. 3). Owing to the destruction of cellmembranes, it is known that more ROS are produced, that is,strong biophoton radiation occurs. In the case of 1 M, on theother hand, plasmolysis occurred slowly, but disruption wasnot observed after 20 min, and in the case of 0.1 M, no mor-phological changes were discerned. Thus, it becomes obvi-ous that disruption of cell membranes are accompanied withextraordinarily strong photon radiation. We will discuss thisagain later with additional results.

3.2 Growth stage dependence of photon emissionThere are many growth stages in the germination process,

such as the development of radicle, hypocotyl and cotyledon.When these occur, plant hormone levels in the roots change,because some morphological and metabolic changes are ledby the endogenous hormones.17) Since plant hormone can actas a stress messenger,17) the salt resistibility of roots mostlikely depends on their growth stages. In order to examinesuch different responses to salt, we measured the responses attwo different growth stages, 48 h and 96 h, corresponding tothe time when the maximum acceleration rate and the maxi-mum velocity of root elongation are respectively achieved.

Figure 4 shows the biophoton responses of roots to the saltstresses at different growth stages. In the case of the trigger-ing time tg = 48 h at which the sample is soaked with NaClsolution, the photon intensity in the case of 0 M continues toincrease fort ≥ 48 h in the same way as in the case of normalgrowth. In 0.01 M, 0.1 M and 1 M-NaCl treatment, however,photon intensity decreases compared with the reference. Thissuggests that these decreases resulted from growth inhibitionand physiological damage in roots. In the case of the trigger-ing time tg = 96 h, on the other hand, the photon intensity

Time [h]0 36 84 132 180

30

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5Ro

ot L

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gth

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]

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Fig. 1. Growth of root: Fromt = 0 to 72 h in pure water, after 72 h inNaCl solutions. solid line: reference, closed box: 0.01 M-NaCl, closedcircle: 0.1 M, open circle: 1 M.

Table I. Salt tolerance of red beans.

NaCl contentration Impression Evaluation of(M/l ) damage degree

0.01surviving

physiologigal differences from ref-erence were barely visible mild

0.1serious damage occurred (root capchanged color) strong

1lethal

protoplasm effusion lethal

4.5 critical

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nt [%

]

Fig. 2. Photon responses after soaking in various NaCl concentrations att = 168 h. Thin solid line: 0 M-NaCl (reference), short broken line:0.01 M-NaCl, dash-dot line: 0.1 M, long broken line: 1 M, thick solid line:4.5 M. Stressed time indicates the development over time since the rootswere soaked in salt solution att = 168 h.

Jpn. J. Appl. Phys. Vol. 39 (2000) Pt. 1, No. 6A T. OHYA et al. 3697

in 0.01 M-NaCl treatment continues to increase similarly tothe reference, while in the case of 0.1 M and 1 M, their in-tensity decreases. These suggest that roots of triggering timetg = 48 h are more affected than attg = 96 h by 0.01 M-NaCl. In order to investigate the growth stage dependence ofsalt sensitivity, we compared a root grown in 0.01 M NaCl so-lution from the beginning (tg = 0 h) with that fromtg = 72 h.The final length of roots soaked in NaCl solution oftg = 0 his shorter than that oftg = 72 h, as shown in Fig. 5. Thus,it became clear that roots at an early growth stage are moresensitive to salinity than at a later growth stage.

3.3 Spatial distribution of photon emissionWe measured the spatial distribution of photon emission

from a root under salinity to determine the region where pho-tons are strongly emitted. Figure 6 shows two-dimensionalimages of biophoton radiated from a root under 4.5 M salin-ity. The summation is performed for 2.5 h. The upper sideand lower side of this figure correspond to the root cap andbase, respectively. The part of the root with strong radiationappeared at a root apex about 15 min after NaCl treatment,and expanded toward the base of the root and gradually de-cayed as time proceeded. This shows that the salt resistibilityof a root by salt stress varies depending on the location of theroot. Moreover, it is well known that there are many imma-

ture cells such as cells of apical meristem near a root cap, andthey have poor mechanical and chemical tolerance.19) There-fore, in this region the progress of cell destruction due to saltstress may be faster than in the other regions. This is probablyone reason for the observation that the photon emission at aroot apex is more rapid and stronger than in other parts.

3.4 Oxygen dependence of photon emissionROS is one main source of biophoton emission,9–11,13,20)

which is generated not only from metabolism but also stressreaction.8) Therefore, an increase of photon intensity is ex-pected after NaCl treatment as a result of additional ROS gen-eration in plants. However, the experimental results describedabove show unexpected responses, that is, photon intensitydecreases after NaCl treatment at concentrations of 0.01 M,

3698 Jpn. J. Appl. Phys. Vol. 39 (2000) Pt. 1, No. 6A T. OHYA et al.

tg = 96 h

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]N

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Fig. 4. Photon intensity change by application of various NaCl concentra-tions at different growth stages; (a) 48 h, (b) 96 h. Thin solid line: 0 M(reference), short broken line: 0.01 M-NaCl, dash-dot line: 0.1 M, longbroken line: 1 M. Stressed time indicates the development over time sincethe roots at each growth stage were soaked in salt solution.

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]R

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0.010.01 (72h)

0.10.1 (72h)

Fig. 5. Growth inhibition of NaCl concentration and growth stage depen-dence of the inhibition. (a) 0.1 M-NaCl, (b) 0.01 M-NaCl. Open circleand open box indicate the growth in NaCl solution from the beginning for0.1 M and 0.01 M, respectively. Closed circle and box indicate the growthin NaCl solution fromt = 72 h for 0.1 M and 0.01 M, respectively. Solidline: 0 M-NaCl (reference).

0.1 M and 1 M. In order to clarify this, the change of inten-sity related to ROS in the whole photon emission should bemeasured. In order to classify the biophoton emission as be-ing of ROS origin or not, we measured photon intensity uponexchanging nitrogen for air in various NaCl concentrations(Fig. 7). Figure 7 shows that the photon intensity decreasesat all NaCl concentrations as a result of the gas exchangefrom air to nitrogen. The decrease at 4.5 M was the greatestamong the four concentrations, and the decreases at 0.01 Mand 0.1 M were almost equal, both of which were greaterthan that at 0 M. Though the decrease varies with the NaClconcentrations, universal aspects are drawn schematically inFig. 8. Ia(x) and In(x) are the photon intensity of a sample atx M-NaCl stress in air and nitrogen, respectively. Then, thedecrease of the photon intensity due to the gas exchange canbe regarded as the photon emission which depends on oxy-gen. Therefore, we defined the oxygen-dependent componentIo as eq. (2).

Io(x) = Ia(x)− In(x) (2)

Here,In is the intensity originating from anaerobic reparation.In the case of 4.5 M, the photon intensityIn(4.5) in nitro-

gen was greater than the referenceIn(0). Namely, the photonemission which was independent of oxygen increased owingto the salt stress. The difference in the photon intensityIa(4.5)andIn(4.5), i.e., Io(4.5), was greater than the referenceIo(0).This shows that the oxygen-dependent photon emissionIo

also increases owing to the salt stress. Moreover, the increaseof the oxygen-dependent photon emission is much greaterthan the oxygen-independent one. Therefore, this suggeststhat the extraordinary photon radiation is mainly caused bythe increase in the oxygen-dependent componentIo. In thecase of 0.01 M and 0.1 M, the photon intensities under nitro-gen atmosphere, i.e.,In(0.01) and In(0.1), were weaker thanthe reference. Namely, the oxygen-independent photon emis-sion decreased by the application of the salt stress. In the caseof 0.01 M and 0.1 M, however, theIo increases because the

Fig. 3. Micrographs of root cells. (a) a 4.5 M-NaCl solution drop was added, (b) a 1 M-NaCl drop was added. The big open arrowsindicate plasmolysis (4.5 M, 1 M) and broken cell membrane (4.5 M only). Temporal changes of cells do not directly correspond toresults obtained from photon intensity change since here plant cells are bare and immediately feel strong stress.

Fig. 6. (a) Biophoton in a two-dimensional image from a root under NaCl stress. The summation of photons was done for 2.5 h. Here,an inset bar shows 1 mm length in the image, and the location of strong emission is the growing point near the root cap. (b) Temporalchange of intensity of total biophoton. The intensity was the summation of the counts-per-second at the bright area in (a).

Jpn. J. Appl. Phys. Vol. 39 (2000) Pt. 1, No. 6A T. OHYA et al. 3699

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didates for new sources of the ROS generation. One is a hy-persensitive response which is the self-defensive mechanismof plants.13) The other is the degradation of cytosol by someenzymes such as oxidase and amine oxidase leaked from theorganelles whose membranes were broken by salinity.10,14)

However, there is still room for argument on these processesand more study is required.

4. Concluding Remarks

We have studied the photon response of red beans undersalinity condition. It became clear that photon intensity ofred beans due to salt stress depends not only on the NaClconcentration but also the application time. Thus, photon re-sponse shows the temporal change of salt tolerance in plantsaccording to their growth. The salt stress induced an increasein the oxygen-dependent photon emission. It suggests thatROS generation in the cell is activated by the salt stress. Asa result, it shows that photon radiation from plants is linkedto physiological change via the endogenous ROS level undersalt stress. Therefore, it seems that biophoton measurementcan be used to estimate the salt damage of plants in the earlystages.

Acknowledgements

This work is partially supported by the Grants-in-Aid forScientific Research (No. 09878108) from the Ministry of Ed-ucation, Science, Sports and Culture.

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decrease of photon intensity due to the gas exchange is greaterthan in the reference, though their net intensity was compara-ble. Consequently, it becomes obvious that salt stress leadsto an increase of photon emission which depends on oxygen.This suggests the increase of endogenous ROS in plants; wewould need to examine the photon response in various saltconcentrations under nitrogen atmosphere to understand thisrelationship quantitatively.

Based on the present results, we discuss the causes of theextraordinary photon radiation at 4.5 M. It is known that only0.3 M-NaCl causes nuclear and DNA degradation in a root ofbarley seedling.21) Thus compared to the case of the 0.3 M-NaCl solution, in 4.5 M-NaCl the damage may extend notonly into cell membranes but also into other organelles. Insuch a case, the regular route to the ROS generation systemwill not work; for example, an electron transport chain in mi-tochondria may be blocked. Then, we may propose two can-

Pho

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TimeFig. 8. Schematic drawing of photon intensity change observed in Fig. 7.

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Fig. 7. Photon intensity in nitrogen for various NaCl concentrations. At-mosphere changed from air to nitrogen att = 10 h. Thin solid line: 0 M(reference), short broken line: 0.01 M-NaCl, dash-dot line: 0.1 M, thicksolid line: 4.5 M.