Biophoton Emission Due to Drought Injury in Red Beans:

Possibility of Early Detection of Drought Injury

Tomoyuki OHYA1, Satoshi YOSHIDA2, Ryuzou KAWABATA1, Hirotaka OKABE1 and Shoichi KAI1

1Department of Applied Physics, Faculty of Engineering, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan2Biotron Institute, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

(Received October 12, 2001; accepted for publication March 27, 2002)

We study biophoton emission from red beans (Vigna angularis) during germination and seedling stages under drought stress.Strong photon emission is observed at the root apex when the beans are subjected to the dry condition. The spatial distributionof the emission is broader than that of emission due to the application of strong salt stress reported previously [T. Ohya et al.:Jpn. J. Appl. Phys. 39 (2000) 3696]. When they are rewatered, strong photon emission from them is again observed. As theirdrought damage is weaker, the intensity of the photon emission is weaker. Photon emission from damaged roots indicates theirphysiological response to external stress, that is, photon emission intensity measurement is useful for detecting physiologicalchanges and evaluating the degrees of such changes before serious damage takes place without any invasion anddestruction. [DOI: 10.1143/JJAP.41.4766]

KEYWORDS: biophoton emission, reactive oxygen species (ROS), drought stress, damage estimation, Vigna angularis

1. Introduction

Serious problems persisting in agriculture include disas-ters such as flooding, salinity, chilling and freezing. Forhuman survival in the future, such problems must be solvedby developing advanced science and technology. Drought isone of the most serious and broadly occurring disasters inthe world. In such areas, one cannot preserve enough waterfor agriculture. A few years ago, for instance, inferioragricultural products were obtained from corn-producingareas in the US due to serious water shortage that wasinduced by the lowering of the underground water level.Thus, the total produce is affected by the amount of watersupply.

In order to avoid poor crop production due to insufficientwater supply, it is necessary to control the amount of waterused for cultivation all year round, that is, saving water is themain subject. To avoid damage caused by lack of water, onemust detect and grasp as early as possible physiologicalchanges in plants caused by water stress. To establish aneasy and precise technique for monitoring water balance,therefore, we focus on spontaneous biophoton emission as asignificant signal from plants undergoing physiologicalchanges.

Extremely weak bioluminescence, the so-called biophoton(BP), is the spontaneous light emission from livingorganisms.1,2) BP intensity is much weaker than otherspecific bioluminescence such as that from a firefly. Theorigin of the BP is some reactive oxygen species (ROS) andother radicals produced by various biochemical reactions tomaintain homeostasis and respond to environmentalstress.3,4) Strong BP emission, for example, was observedfrom wound stress (a kind of mechanical stress) in planttissue, and singlet oxygen production in plant cells throughphenolic peroxidation was found.5,6) Similarly, BP emissionfrom a drought-stressed plant has also been reported,7,8) butno quantitative measurements had been carried out so far.

Thus, for plants under mechanical and/or chemicalstresses such as heat shock, chilling, wound, or pH change,the BP emission from their roots and leaves often drasticallychanges.5,6,9–12) These emissions are, as described above, aresult of ROS generation due to metabolic disorder and the

self-defense mechanism called hypersensitive reaction. Weare therefore interested in BP as a means of obtainingsignificant information related to the internal physiology ofplants and of using it as a control signal for plant growth. Inorder to use it appropriately, we must understand thecharacteristics of the BP emission from plants. Within theframework of early damage detection, we have reported BPintensity changes due to various externally applied stres-ses.13,14)

The results obtained in our previous studies, revealed thatdamage due to salt stress enhanced internal ROS generation,which is an origin of BP radiation. We showed that BPmeasurement is useful for evaluating physiological damagein plants due to salt stress.13,14) In the same manner, weexpect that new information of the mechanism of droughttolerance and damage can be acquired through BP measure-ments.

In the present experiment, as our first step, we detect thephysiological changes during germination and seedlingstages in red bean roots due to drought stress. We measureBP emission from the root and the length of root under drycondition in order to know the stress response of a plant.Then, we investigate the dynamical change of the BPemission intensity when drought stress is applied andremoved by rewatering. Optical microscopic observationof plant cells is simultaneously carried out to monitor themorphological changes caused by the stress.

2. Experimental

Red bean seeds (Vigna angularis) were chosen such thatthey be within the standard deviation of their weightdistribution (the total number of seeds was 3,000) in orderto eliminate individuality of each seed as much as possible.We simultaneously prepared two sampling groups fromthem, because two different measurements for all experi-ments, namely root length and photon emission measure-ments, were carried out simultaneously. These two groupswere placed in an incubator for 24 h under conditions ofhumidity RH ¼ 95% and temperature T ¼ 35�C to inducegermination. After 24 h, they were transferred into a growthchamber (Shimadzu BEC II-350) with RH ¼ 82%, T ¼24�C. We assume this to be the time origin t ¼ 0 hereafter.

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#2002 The Japan Society of Applied Physics

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The entire culture process of the samples was carried outin a dark room to avoid photosynthesis. Only whenmeasuring the root length every 12 h did, we illuminate avery weak green light whose intensity was lower than theminimum light intensity for inducing photosynthesis.Twenty roots were simultaneously grown in a petri dishfor root length measurement. Pure water (conductivity:0.10�S) distilled and filtered by a purifier (Advantec Co.,AQUARIUS) was used for all experiments. The culturesolution was changed every 12 h to avoid contamination.

2.1 Biophoton measurementA universal photon counting system (Hamamatsu Photo-

nics C-2550, photomultiplier R649, spectral response range300–850 nm) and a two-dimensional photon counting system(Hamamatsu Photonics PIAS-TI500 and C-1809) were usedfor measurements of the temporal changes of total BP countsand their spatial distributions, respectively. All photon-counting systems were set in an iron box to shield them fromelectric and magnetic noises. The details of the photonmeasurements have been described previously.13,15) Beforestarting the photon measurements, an inoperculate petri dishwas placed in the dark room for several hours to avoidluminescence from itself.

2.2 Microscopic observation of root cellsThe sample for microscopic observation was prepared as

follows: A sample root was washed by water and tenderlyblotted with soft paper to remove excess water. Then, it wasembedded in agar solution. After cooling at gelationtemperature, and a sample 150–250�m thick was slicedoff by a microslicer (Dozaka EM DTK-1500). This slicedsample was mounted on a glass slide and observed by anoptical microscope (Nikon Optiphoto-2).

3. Results

3.1 Root growth and drought injuryAs shown in Fig. 1, the final root growth was shorter as

the drought period tD became longer. When the sampleswere stressed from t ¼ 48 h (i.e., ts ¼ 48 h), the roots thatwere rewatered after 1 and 2 h (i.e., tD ¼ 1 and 2 h)continued to grow without suppression. As shown in Fig.2(b), no morphological change could be observed in theoptical microscopic image (tD ¼ 1 h). In the case ofseedlings rewatered after the drought period of 6 and 12 h(i.e., tD ¼ 6 h and 12 h), a considerable percentage (e.g.,typically 25% in 40 seeds) stopped growing. The remaining75% exhibited root growth but were much shorter than thereference. In these cases, optical microscopic observationrevealed that the root apex changed color from white tosemitransparent brown, and cell walls on the root surfacewere remarkably deformed [see Fig. 2(c)]. (The micrographfor 6 h is not shown here because it is similar to that for12 h.) Some lateral roots grew from the root base afterrewatering while the main roots did not. In the case ofseedlings rewatered after the drought period of 36 h, bothmain and lateral roots did not grow further. Morphologicaland color changes of the root apex, which, as describedabove, indicated serious damage, were clearly observed in60% of the samples. In optical microscopic observation, thedeformation of cell walls and the collapse of cells were

observed, cell membranes of the roots were ruptured, andcytoplasm exuded into the culture solution.

At ts ¼ 96 h, no root elongation was observed in someseedlings after rewatering, even when the drought stress wasless than 1 h. The percentage of surviving roots at ts ¼ 96 his lower than that at ts ¼ 48 h.

3.2 Biophoton emission due to drought injuryFigure 3 shows temporal changes of the BP count of red

bean seedlings under the dry condition (T ¼ 24�C RH ¼78� 5%) at various growth stages, t ¼ 48 h, 96 h and 168 hcorresponding to the stages of the maximum accelerationrate, the maximum speed, and the start of root growthsaturation. The vertical axis shows normalized excessphoton count I defined by eq. (3:1).

I ¼X

X0

� 1

� �� 100; ð3:1Þ

where X0 (count per hour: cph) is averaged for 5.5 himmediately before the application of drought stress, and X

is photon count under the dry condition. At ts ¼ 48 h, BPintensity is immediately enhanced by the application ofdrought stress, then decreases quickly to reach a minimum ataround 120min. With time, the intensity slightly increasesagain and shows a flat peak at 10 h. In contrast, at ts ¼ 96 hand 168h, after a sharp increase the BP intensity decreases,reaches a minimum around 3–5 h (¼ t2) and increases again.Thus, the time to reach the minimum point in the latergrowth stage becomes longer than that in the earlier growthstage. The common tendency in the BP temporal change of

Fig. 1. Influence of drought stress on root growth. (a) Root length: Arrows

denote the start of drought stress. ts denotes the start of the drought

period. (b) Application of drought stress at ts ¼ 48 h: Here tD denotes the

period of drought stress application. The total number of samples usedhere is 140 (20 seeds for each). Bars indicate standard errors.

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Fig. 3 is shown schematically in Fig. 4. Here we can definethree characteristic time points, t1, t2 and t3, correspondingto the times when the BP intensity exhibits the firstmaximum point, the first minimum point and the secondmaximum point, respectively.

3.3 Two-dimensional photon images of stressed rootTwo-dimensional BP emission from the drought-stressed

root (T ¼ 24�C RH ¼ 50� 5%) was studied in order tounderstand the origin and the location of strong photonemission. Samples in the intermediate growth stage t ¼ 110–140 h were chosen. Figure 5 shows two-dimensional imagesof BP emission from a stressed root, where photon countingof each image was done for 1 h. As seen in the images, whilephotons are essentially emitted from the entire root, theintensity is stronger from the root apex than from the otherparts. The intensity of photons emitted from the root apexdue to the application of drought stress reaches a maximumwithin 30min, and gradually decays over the subsequent 5 to6 h, as shown in Fig. 6.

3.4 Photon response on applying and removing droughtstress

Strong BP radiation was induced when drought stress wasapplied and rewatering was performed, as shown in Fig. 7.Figure 8 shows the comparison of the photon intensitybetween the first peak (P1) and the second peak (PR), forbringing a root in the dry condition and in the rewatered one.Here the shaded area indicates the drought period tD. Theheight of PR is always greater than that of P1, which dependson tD, and becomes maximum at about tD ¼ 20 h (Fig. 8).

4. Discussion

As already described, when the drought period tD waslonger, the final root length was shorter, that is, the finallengths of the stressed roots were always shorter than thereference roots, and the dispersion of drought resistibilitywas considerable. At ts ¼ 48 h, a considerable percentage ofseeds were seriously damaged in the longer drought periodthan 6 h. Their root apexes become discolored, and cell wallson the surface of the root are remarkably deformed [see Fig.2(c)]. Regarding the color change of the damaged roots,

Fig. 2. Micrographs of the slices at 2–5mm from the root apex of seminalroots after rewatering. Seedlings were subjected to dry conditions at

ts ¼ 48 h for (a) 0 h (reference), (b) 1 h, and (c) 12 h.

Fig. 3. Temporal change of BP count of sample seedlings at various

growth stages. Each group of samples (20 seedlings) was set under the dry

condition at various growth stages: ts ¼ 48 h (dotted line), ts ¼ 96 h (solidline) and ts ¼ 168 h (short-broken line). tD is the drought period when the

sample seedlings are maintained under the dry condition.

Fig. 4. Schematic drawing of the temporal change of the BP intensity

observed in Fig. 3.

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lignification of cortical cells may be induced by waterdepression or mechanical deformation of the tissues.16)

Moreover, the development of lateral roots indicates thatcells at the root apex are less robust than cells at the rootbase. At ts ¼ 96 h, no root elongation was observed for someseedlings after rewatering, even when the drought periodwas shorter than 1 h. The drought damage at ts ¼ 96 h isobviously more serious than that at ts ¼ 48 h, because thepercentage of surviving roots at ts ¼ 96 h is lower than thatat ts ¼ 48 h. The reason can be explained as follows. Thedehydration process at ts ¼ 96 h is faster than that atts ¼ 48 h since a seed acts as a water source for its root.The root length at ts ¼ 96 h is larger than that at ts ¼ 48 h.Therefore, the larger root length is disadvantageous forwater transport from the seed, and causes more seriousdamage at ts ¼ 96 h.

The intensity of BP emission is obviously correlated to theinhibition of root growth, namely, the intensities of peaks P1

and P2 depend on the growth stage, as shown in Fig. 3.When stress was applied in the intermediate growth stage

t ¼ 96 h, root damage is more serious than that of thereference as shown in Fig. 1, and the intensities of the twopeaks are strongest compared to the other growth stages. Thegrowth-stage dependence of external stresses has beenalready reported.14,17) According to the results obtainedthere, the stress applied at the earlier growth stage had agreater influence. Similarly, when stronger BP intensity wasobserved, root growth was inhibited more seriously, that is,BP radiation in the dry condition reflected the degree ofdrought damage. As described previously, the dehydrationprocess was faster in the later growth stage of roots, that is,the faster dehydration leads to more serious drought damage.Therefore, the actual drought damage (i.e., the BP intensity)depends on the competition between growth-stage dependentstress sensitivity and length-dependent dehydration.

Let us consider here the mechanism of BP emission fromthe drought damage. When oxidative decomposition oflipids is induced by oxygen in air due to the strong droughtstress, radical chain reactions that produce ROS arepromoted.18) ROS is produced in the ruptured membraneand the cells destroyed by rapid drying. Furthermore, thewater loss of living cells induces various homeostaticchanges, such as the relationship between intra- andextracellular water (the so-called water relation in botany),the physiological function of the membranes and cytoplas-mic metabolic processes that also produce ROS.19) Asdescribed above, BP emission results from these physiolo-gical and structural changes of the cells through exergonicreactions such as direct oxidation by endogenous ROS andautoxidation.20–22)

Furthermore, the BP measured here is the summation ofphotons from the entire seedling including root, seed andshoot. Each part responds to the drought stress with adifferent delay time, and the reason why two peaks and onevalley are observed in the temporal change of BP intensity asschematically shown in Fig. 4 is related to the radiation fromdifferent parts. The first peak P1 is due to the strong BP

Fig. 5. BP images from root apex under the dry condition in the growth stage at 132 h. For each image, BP was accumulated for 1 h.

Here, the inset bar shows 1mm length in the image. The upper and lower parts of the images correspond to the root cap and base,

respectively.

Fig. 6. Temporal change of BP count in apical area A shown in Fig. 5.

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emission from the root apex as shown in Fig. 6, and thesecond peak P2 may be due to that from the root base. Thefirst peak P1 is therefore related to drought damage on rootelongation, since cell division occurs only at the root apex.The reason why BP emission from the root apex is strongerthan that from the base is that the cells in the apex areyounger and have mechanically weaker cell walls23) that arebroken more easily than the old ones near the base. Then, thestronger BP emission from the apex is observed due todestruction of those cells by the stress. To induce furtherelongation after stress application, rewatering should beaccomplished before the destruction of the cells at the apex,that is, t ¼ t2 is the time limit to avoid serious droughtdamage. Such a critical time can be explained as follows.Living systems consist of many complex materials such asproteins, polymers and gel networks. It is well known thatthe dehydration process of such complex materials com-prises two steps: the dehydration processes of free water andbound water.24) Free water is dehydrated as a fast process atthe beginning while bound water is a very slow process.Bound water is tightly bound to biological materials such asproteins, enzymes and membranes. Therefore, if thedehydration process of bound water from such materialsoccurs, the organisms suffer from critical damage and notrecovered again even after rewatering. Thus, time t2 may bethe starting of time the dehydration process of bound water.

The two-dimensional photon image taken near the rootapex showed that the response to drought stress was differentfrom that to salinity, because under high salt stress, strongBP radiation localizes only near the root cap (apex)compared to the drought stress case.14) This may be due todifferences in damaged organs and mechanisms, that is, saltstress is a complex stress composed of osmotic and ion

Fig. 7. Successive BP images during drought and rewatered conditions in the growth stage at 142 h.The BP intensity at root apex

increased on application of drought stress and on rewatering. For each image, BP was accumulated for 1 h. Here, the inset bar shows

1mm length in the image.

Fig. 8. Temporal change of BP emission intensity. (a) The temporal

change of the BP emission intensity during application of drought stress

(shaded area) and after rewatering (see also Fig. 7). (b) Relationshipbetween the drought period and the BP emission intensity PR at

rewatering.

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stress. The root apex tissue comprising many immature cellsis rapidly affected by salt stress, because ions can passthrough the cell walls easily and react with the organism. Incontrast, in the case of drought stress, tissue damageproceeds slowly because the cell wall acts as a barrieragainst for drought.

Strong photon emission was observed on tissue dehydra-tion and the rehydration (see Figs. 5 and 7). The reason whystrong radiation PR appears is considered as follows. Due toserious drought damage under the dry condition for 20 h(t ¼ t3), membranes, cell walls, and vacuoles are disruptedand their contents flow out. Some enzymes (e.g., oxidase,peroxidase and lipase) restimulate chemical reactions byrewatering, and this process may produce excess ROS also.All these exergonic reactions may therefore be sources of theBP emission at PR.

5. Concluding Remarks

The physiological response and the temporal change ofbiophoton emission intensity during the seedling stage werestudied in detail under application of drought stress. In thepresent study, the following findings were obtained: 1)Drought damage induced BP emission from root. 2) Thetemporal change of BP emission intensity depended on thegrowth stage of seedlings and their physiological response.3) The spatio-temporal properties of the drought-induced BPemission were different from those of salinity-induced BPemission. 4) In the rewatered case, a stronger BP emissionthan that due to stress application was also observed. As aconclusion, photon measurement is useful for the earlydetection and quantitative estimation of drought damage aswell as the distinction of stress.

Finally, we would like to mention that based on thepresent study, the photon measurement system can beapplied not only for quick and continuous monitoring ofphysiological changes in plants but also for optimum controlof drought stress, for example, for high-quality cultivars likesweet melon that always requires optimum water stress.Such sophisticated system is not available to date.

Acknowledgement

This work was partially supported by Grants-in-Aid for

Scientific Research from the Ministry of Education, Culture,Sports, Science and Technology.

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