ORIGINAL PAPER

Spontaneous ultra-weak light emissions from wheat seedlingsare rhythmic and synchronized with the time profileof the local gravimetric tide

Thiago A. Moraes & Peter W. Barlow & Emile Klingelé &

Cristiano M. Gallep

Received: 20 March 2012 /Revised: 20 April 2012 /Accepted: 27 April 2012 /Published online: 26 May 2012# Springer-Verlag 2012

Abstract Semi-circadian rhythms of spontaneous photonemission from wheat seedlings germinated and grown ina constant environment (darkened chamber) were foundto be synchronized with the rhythm of the local gravi-metric (lunisolar) tidal acceleration. Time courses of thephoton-count curves were also found to match thegrowth velocity profile of the seedlings. Pair-wise anal-yses of the data—growth, photon count, and tidal—bylocal tracking correlation always revealed significantcoefficients (P>0.7) for more than 80% of any of thetime periods considered. Using fast Fourier transform,the photon-count data revealed periodic components sim-ilar to those of the gravimetric tide. Time courses ofbiophoton emissions would appear to be an additional,useful, and innovative tool in both chronobiological andbiophysical studies.

Keywords Biophoton emission . Chronobiology .

Germination . Gravimetric tide

Introduction

Daily and monthly lunar rhythms are traditionally acknowl-edged by agricultural and forestry communities all over theworld (Kollerstrom and Staudenmaier 2001). They are heldto be key tools in determining the best times not only for thegermination of plant seeds and subsequent harvesting of thecrop but also for ensuring optimal quality of the final plantproduct. This applies not only with respect to food manage-ment, where water percent in plant fruits and leaves oftenaffects their flavor, maturation, and storage (Kollerstromand Staudenmaier 2001), but also for the management oftree wood quality (Zürcher 2001). With respect to this last-mentioned quality, the mechanical properties of timber havebeen found to be strictly correlated to the water content ofwood at the time of tree felling (Zürcher et al. 2010); thisbiological variable has been shown to be modulated inaccordance with the phase of the Moon.

In consideration of possible lunar effects on biologicalmaterials and processes, recent studies have shown that bothplants and animals, in addition to having cycles of activityand inactivity synchronized in relation to the diurnal light/dark patterns of day and night, also show behavioral featuresthat relate to the continually varying (but predictable) posi-tion of the Moon relative to the Earth and the Sun, i.e., thelocal lunisolar tidal rhythm (Klein 2007; Barlow and Fisahn2012). Impressive data, first presented in 1998, indicatedthat the diameter variation of a tree stem (Picea abies)varied in accordance with the local lunisolar tidal cycle(Zürcher et al. 1998), and further analysis has uncoveredsimilar corroborative evidence as well as additional indica-tions of a temporal correlation with geomagnetic activity(Barlow et al. 2010), which may itself be governed by thelunar tide. In addition, tidally modulated rhythms have beenfound for the imbibition and germination of bean seeds

Communicated by: Sven Thatje

T. A. Moraes :C. M. Gallep (*)School of Technology, University of Campinas,Rua Paschoal Marmo 1888,13484-332 Limeira, SP, Brazile-mail: gallep@ft.unicamp.br

P. W. BarlowSchool of Biological Sciences, University of Bristol,Bristol BS8 1UG, UKe-mail: p.w.barlow@bristol.ac.uk

E. KlingeléInstitute of Geodesy and Photogrammetry, ETH, HIL D 42.2,Wolfgang-Pauli-Strasse 15,8093 Zurich, Switzerlande-mail: klingele@geod.ethz.ch

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(Brown and Chow 1973; Spruyt et al. 1987) as well as thenastic movements of bean leaves (Barlow and Fisahn 2012;Barlow et al. 2008).

“Biophoton” measurements—photon-counting experi-ments in which weak spontaneous light emissions from non-luminescent organisms are recorded—is a prominent tool forreal-time inference of organism activity, especially in caseswhere minimal disturbance to the biological material is re-quired (Kobayashi and Inaba 2000). This biophoton emission,which is also known as “spurious chemiluminescence” or“ultra-weak photon emission (UWPE)”, is a phenomenonrelated to a living organism’s exogenous and endogenousconditions (Slawinska and Polewski 1992), and was discov-ered to take place during seed germination by Colli andFacchini (1954). Subsequent studies have linked photon emis-sion with events occurring at the cell membrane and in stress-ful circumstances, in particular, may also involve theoxidation of lipid species (Birtic et al. 2011). Colli et al.(1955) also showed that the amount of light emitted in thevisible spectrum was directly related to singlet oxygen (1O2)production within the biological material and correlated withthe vigor of the initial seed sample. As a result, UWPE fromseeds has been employed as a rapid means of evaluating seedquality and viability. For cereal seeds, for example, it ispossible, after only a fewminutes of imbibition, to distinguishthose seeds which will produce stronger sprouts upon germi-nation from those which will be weaker (Chen et al. 2003).Then, during the subsequent growth period, the whole of eachseedling emits light, the source of the luminescence being theradicle, the cotyledon, and the endosperm, the latter tissue,perhaps because of its greater mass and rapid catabolic me-tabolism, producing the greatest signal (Chao 1998). Further-more, slightly injured plants often present higher photonemissions than do undamaged and intact plant material, asfound for maize leaves, for example (Yoshinaga et al. 2006).More damaging types of stress, such as exposure to toxicchemicals, in addition to diminishing seedling development,also reduce the total light emission. Summarizing many yearsof work involving biophoton measurements, we have foundthat the collection of such data is a useful predictive tool,providing a routine means of distinguishing between stocks ofseeds which would later show either high or low rates of seedgermination. Predictive patterns of cellular light emission is auseful diagnostic tool in an agricultural context by way ofidentifying plant biotypes with differences in herbicide sensi-tivity (Inagaki et al. 2009), as well as in the medical sciences(Takeda et al. 2004).

Biophoton emission also displays a circadian rhythm.Whereas this is characteristic of non-stressed samples (Gallepand dos Santos 2007), stressed samples show diminishedrhythmicity. Spontaneous circadian rhythms of photon emis-sion were first described for seedlings raise in darkness byYan (2005), and since this first discovery, more than 9,000 h

of biophotonic tests have been performed in our laboratory(LaFA/FT—Unicamp), using wheat seedlings as the test ma-terial. Not only were daily, circadian-type rhythms routinelyobserved but so also were monthly rhythms. A first publica-tion on these observations appeared in a conference in 2007and was related to two simultaneous series of germinationtests (Gallep and dos Santos 2007).

In the present work, a detailed analysis has been made ofthe daily rhythm of spontaneous light emission from wheatseedlings raised in a controlled, dark environment to try andestablish its source. Our main finding is that the photonemission from the seedlings appears to vary in synchronywith the estimated local lunisolar acceleration (i.e., thelunisolar gravitational tidal cycle at the experimental site)and its first derivative (tidal velocity). Coincidence betweenthe respective time courses of the photon count and the locallunisolar tide has been assessed statistically using Pearson’slocal correlation coefficient, as well as by a fast Fouriertransform (FFT) and coherence analysis to determine simi-larities in periodic time components. We have also placedthese results of spontaneous photon emission in the contextof lunar-related monthly and seasonal periodicities.

Materials and methods

The seed stock of bread wheat (Triticum aestivum L., cv.MaisVita) used for the experiments was stored in darkness toavoid induction of any diurnal rhythm. Stocks were changed atleast twice per semester (6 months) to ensure high germinationrate (>90% germinated seeds in 96 h). Prior to each photon-counting test, a set of triplicate samples was checked foroverall germination efficiency (% germination and vigor—i.e., the total seedling length on the fourth day after imbibitioncommenced) according to the standardized Rules for SeedAnalysis (RSA, Ministry of Agriculture, Brazil; p. 365,1992). Then, 50 wheat seeds were chosen at random fromthe seed stock and placed in a Petri dish (10-cm diameter).Imbibition and germination took place on filter paper moist-ened with 10 mL of double distilled water. Each test alwayscommenced at the same time of day, 09:00 h. Seeds forgermination and photon counting were handled under minimaland constant illumination in order to avoid not only prolongedsubsequent luminescence from the filter paper and the waterremaining from imbibition but also the possibility of entrain-ment of growth or photon-emission rhythms. They were alsomaintained in darkness before, during, and after transfer to thephoton-counting chamber.

Photon counting was performed in a dark chamber withcontrolled temperature (21±1°C) using a dedicated photo-multiplier tube (Hamamatsu H7360-01) with lowbackground-count noise (<170 counts per 10 s from an emptychamber) and with sensitivity to light in the visible range

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(Gallep 2005). The photon count was recorded in 10-s inte-grating windows and commenced just after the start of sampleimbibition for series 1 and 3 (test V1, see below) or after afurther 24 h of incubation in darkness for series 2.

From 2007 up to 2011, more than 250 germination tests,spanning more than 4,000 h in non-stressing (control) and5,000 h in stressing conditions, were undertaken and therecords of the respective time courses of biophoton emissionwere stored in digital form and available at the authors’website (http://www.ft.unicamp.br/∼gallep/Gerais/todas.pdf).Here, we present details of biophoton counts retrieved fromthis data store which concerns two representative germinationtests performed during 2008.

Series 1 is comprised of six 3-day germination testsperformed during March 2008 (raw data at Gallep and dosSantos 2007). Series 2 is comprised of 22 2-day tests per-formed during January and February 2008. A further seriesof tests, series 3, combining photon counting and simulta-neous video recording of a duplicate sample of seeds underlow, constant IR illumination, was made during 2011. Thesetests of series 3 were performed in order to provide timecourses of shoot elongation which could be related to aparallel counting of photons obtained from a replicate sam-ple of seeds. The results from one representative test (V1) ofa number of such tests in series 3 are presented here.

In test V1, simultaneous video recordings were analyzedfrom single seedlings placed in vertical dishes to determinetemporal variations in the rate of seedling growth, using cole-optile elongation as a convenient measure. These data wererelated to photon counts obtained simultaneously from a rep-licate set of seedlings grown under identical conditions bymeans of a statistical “coherence” function (www.originlab.com/www/helponline/Origin/en/). The video images were ac-quired at intervals of 5.75 min under low intensity infra-red(IR) illumination with a standard video camera in night-shot(IR-enhanced) mode. A total of 1,205 images were gatheredover a period of 115 h 40 min. Based on these images, acompressed 40-s video was created (29 frames/s) from whichthe time course of the coleoptile growth rate for each seedlingcould be analyzed. The video recordings are available online(http://www.ft.unicamp.br/∼gallep/Gerais/t10.mp4).

In order to analyze the data from photon counting, thevariability due to thermal noise was minimized by smooth-ing the counts using local averages [window sizes (w) ofw0100 and w01,000 data points]. The resulting graphs ofthe photon-count time courses showed rising and fallingportions (ramps). Inflection points at intervals along theseramps were identified as positions where there were pro-nounced changes in the first- and second-order derivatives(inclination and concavity) of the curves. These inflectionpoints could then be related to the next closest peak ortrough in the lunisolar tidal time course. Then, in order tovisualize the oscillatory nature of the photon-count data, as

defined by the inflection points, 24-h segments of the pho-ton count graph were selected, and a linear regression ofphoton counts versus time was estimated. The “residuals” ofthe regression, obtained by subtracting the actual photoncount value, PC, from the estimated mean at a given time,provide a time course of the temporal variation of PC(±δPC) over the selected 24-h time period. This residual,±δPC, is analogous to the variation of tree diameter, ±δD,described by Zürcher et al. (1998) and others (Barlow andFisahn 2012; Barlow et al. 2010).

Time courses of the local lunisolar tidal force were estimat-ed for the laboratory site at Limeira SP, Brazil according to theprogram “Etide” developed by the Institute of Geodesy andPhotogrammetry, ETH-Zurich, based on Longman (1959).The Etide program estimates the vertical component of thelunisolar tidal force at any given location and date, and pro-vides an output in terms of the gravimetric variation, δg, at thatthe specified location in units of μGal (9.81×108 μGal01 g ofEarthly gravitational acceleration). These gravimetric unitstherefore stand proxy for the actual lunisolar gravitationalforce. Values of δg are estimated at 15-min intervals over therequired period and are presented on a time base in accordancewith Universal Time (UTC). Hence, the time courses wereadjusted, as were the local times at which the photon countswere collected, to conform to a common time base expressedin terms of the local solar time (UTC −3 h 9 min 41 s) at thelocation of Limeira. Times of rising and setting of Sun andMoon (http://tbone.biol.sc.edu/tide), according to local solartime, were also estimated for this location. Consistency of theEtide estimates for the specific site was also checked bycomparison with official (Brazilian Navy, www.mar.mil.br/dhn/chm/tabuas/index.htm) data for ocean tide peaks at asimilar latitude and nearby longitude. This checks, however,only a very general test of consistency since the timing ofmarine and oceanic tides is subject to many modifying factors(water currents, shore-line topography, etc.).

Local correlation (Pearson) coefficients (Pcoef) curveswere calculated for the smoothed photon-count curve, andrespective lunisolar gravity profile derived from Etide, usingsliding windows as indicated in Papadimitriou et al. (2006).The inflection points can be defined also according to thefirst derivative (rate of increase) of the photon count timecourse. The residuals, estimated as ±δPC, were then com-pared with the tidal variation time profiles as well as theirperiodic components using auto- and cross-correlation func-tions and FFT in order to assess the similarity of theirperiodic harmonics. Similar statistical procedures were alsoapplied to the growth velocities obtained from the videoimages of emerging seedling coleoptiles. In particular, testswere made between the correspondences of periodicities ingrowth, ±δPC (the variation in photon emission), and gravi-metric tidal characteristics such as ±δg and its firstderivative.

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Results

First, we present results (Figs. 1 and 2) of analyses of testV1 from series 3 in which photon counts were collectedfrom the germinating seeds in parallel with a video record ofthe course of seedling growth.

The lower part of Fig. 1 shows the smoothed photon counttogether with its local 24-h linear regression. Also shown arethe simultaneous records of coleoptile elongation in three seed-lings (an image from the video is shown in the inset), and therising and setting times for Sun and Moon. The upper part ofFig. 1 shows the curve of the residuals (i.e., the photon-countvariation, ±δPC) around its local 24-h linear regression, super-imposed upon the tidal profile, ±δg, and its first time derivative.

The photon-count measurement commenced just after thestart of seed imbibition, at 09:00 h on 17/06/2011 (Fig. 1).During the first 16–24 h, a strong but diminishing signalwas recorded. This is due mainly to the emission of lightstored in both the water and the filter paper used for thesetting up of the material. Thereafter, the photon-countcurve showed small increases, commencing at 30 h and42 h. These times coincide with Sun rise and Sun set,respectively (see symbols on the horizontal time axis). Dur-ing this 42-h interval, the profile of the photon countsappears to be synchronized with the gravimetric tidal

profile, the peaks and troughs of the photon count and itsvariation, ±δPC, coinciding, respectively, with the troughsand peaks of the tidal profile, ±δg. The vertical axis of ±δghas been inverted (negative values uppermost). Such nega-tive values indicate a diminished gravity attraction towardsthe Earth. In the remaining interval, post-42 h, inflectionpoints of ±δPC coincide with analogous extremata, or turn-ing points, in the gravimetric profile. These points occuraround 60 h, 73 h, 102 h, 111 h, and 118 h. Observations

Fig. 1 Test V1. Lower panel—smoothed temporal patterns ofthe photon count (using twowindows of either w=100 orw=1,000 counts) from thespontaneous light emittedduring wheat seed germinationand its local (24 h) linear fitting(dotted straight lines). Alsoshown are the coleoptile(“leaf”) lengths for threeseedlings (triangles) whichwere measured in parallel froma video recording. Time of rise/set of Sun and Moon are shownon the time axis. Upper panel—the gravimetric tidal profile andits first time derivativesuperimposed upon the photon-count residuals following locallinear regression. The boxbetween the two panelsexplains the various symbolsused in the graphs. The sametime axis applies to both panels

Fig. 2 Test V1. The fast Fourier transform applied to the gravimetric(δg) and to the residual curves for δPC

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and coincidences of this kind for δPC were regularly foundin relation to δg in many similar tests even when the detailsof the δg profile are changing from day to day. This featurewill be explored further in discussing results gained fromseries 1, where pronounced rises and plateaus are distin-guished in the photon-count data.

The photon-count variation, ±δPC, is always plotted to-gether with the gravimetric tidal profile, ±δg, and its firsttime derivative—the tidal velocity (μGal s−1). In each of thefour intervals selected for detailed analysis, the auto- andcross-correlation functions of these variations and the tidalprofiles were computed (complete data online—http://www.ft.unicamp.br/∼gallep/Gerais/complete1and2.pdf).These functions help to identify similarities between the twoevolving time courses, a property also confirmed by appli-cation of a FFT algorithm.

On many occasions, the photon-count variation, ±δPC,appears to reproduce features of the temporal pattern of thegravimetric tide, ±δg, and to show coincidence with inflectionpoints in that profile, where the tidal velocity is zero. In thefirst 24-h interval (from time 30 to 54 h) δPC is initially flat,indicating photon-count growth of constant velocity. Just afterthe next tidal peak, during the next semi-cycle up to the timeof the next local maxima of tidal velocity, the photon countstarts to decrease (negative values of δPC) in parallel with thedecline in gravimetric tidal profile, δg. From then on, thepattern of δPC accompanies the tidal velocity profile, but witha sudden 90° de-phasing from the original trajectory. Thisindicates an oscillating behavior with strong 6-h semi-cycles.There is also a cryptic, higher order periodicity of 12 and 18 h.Then, during the second 24-h interval (from 54 h to 78 h),±δPC presents a less intense oscillation, with inversion pointscoincident with the extremata of the gravimetric tide and tidalvelocity profiles. During the third 24-h interval (78–102 h),±δPC is strongly periodic, clearly de-phased from the tidalpattern, ±δg, with a further delay during the last quarter period(96–102 h) of this third 24-h interval. The fourth and last24-h interval (102–126 h) presents the most pronounced co-incidence for the ±δPC/gravimetric tide relation, with coinci-dent local peaks and troughs. The corresponding correlationplots present almost equal components around 6 h and 12 h,again with inverted phase relation.

Figure 2 presents the periodic components of both the±δg and ±δPC profiles, obtained by FFT algorithm. In Fig. 3is shown the coleoptile elongation versus photon-countcurve.

It is notable that, in this single test (V1), the ∼12-h and24-h components almost coincide in spectral distributionbut their relative intensities are inverted: the gravimetricdata has stronger components at the 12-h band whereasthose of the photon-count variation, ±δPC, are stronger atthe 24-h band. Although these data represents only asingle test, the synchrony and similarity of the periodic

components from the geophysical and biological variablesare significant, as will be shown below for the tests ofseries 1 and 2.

Coleoptile elongation started to change at the 60th hour,exactly at the time when the photon-count curves started torise more quickly. Seedling growth was always accompaniedby a rise in the photon count, with almost a linear relation, asshown in Fig. 3; remaining oscillations around completelinear growth indicate that not only seedling developmentbut also the accompanying but invisible biochemical processhave been made evident by the spontaneous photon emission.Across the entire data range, the frequency coherence be-tween the elongation and the photon-count curves varied from0.64 to 0.75.

Data from the other test series—1 and 2, in which therewas a large number of successively performed sets of pho-ton counting—are presented in detail online; also includedat this site are plots similar to those done for test V1, as wellas the auto-correlation curves and the local Pearson correla-tion coefficients. Photon counts from series 2 were found tobehave similarly to those described for test V1. There wasstrong, almost monotonic, increase of counts after an initialdecay. However, the subsequent inflection points in thephoton count were not so clear as those in test V1.

Series 2 was run during the summer season, and othertests in summer showed similar behaviors. However, testsrelating to series 1 were performed outside of the summerseason, and these presented pronounced inflections points—i.e., there were better defined rises and plateaus (ramps) inthe photon counts. The Pearson correlation coefficient foreach test from series 1 was always greater than r00.997with respect to paired time points for the photon-countinflexion and the nearest tidal peak. Step-wise increases inthe photon counts were separated by plateaus with similar 6-,12-, and 18-h intervals.

Some selected examples of the δPC curve in coincidencewith gravimetric pattern taken from series 1 are shown inFig. 4a. Remarkable is the first 24-h interval (i.e., the second

Fig. 3 Coleoptile elongation(s) versus the photon count curve (actualvalues)

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day of germination): in all tests, δPC appears as a combina-tion of the gravimetric tide curve with time derivative form-ing a pulse broader than the sinusoidal semi-cycle;nevertheless, it includes the first and the higher periodicharmonics, as can be checked from the correlation functionplots of each test. In all tests, the auto-correlation functionbetween the residuals and gravimetric tides present similarfeatures. This is shown also by the corresponding FFTcurves presented in Fig. 4b. The FFT of δPC presents verysmall 12-h components, which are distinguishable from the18-h component, in coincidence with a weaker band in thegravimetric δg profile. Again, there is a very strong 24-h band almost superimposed upon the corresponding, highergravimetric periodic component.

The next series 2 is especially important since the datawere accumulated in successive trials over a 2-month periodduring the summer season, which ran from January 2 toFebruary 26, 2008, thus extending over almost two lunarcycles (i.e., over four cycles of the tidal amplitude varia-tion). Data of series 2 show distinct synchronism and har-monic (delayed or in-phase) coupling in all but a few cases.Even in less evident cases of oscillation, i.e., with a moremonotonic, linear increase of the photon count, the smallvariations in the δPC profile coincide with singular points ofthe gravimetric δg profile, and so the periodic componentsof both variables are similar, as shown at Fig. 5a in thesuperimposed FFT curves.

In this more extended series 2, the 12-h band is againabsent in the FFT data from δPC, but is present in the δggravimetric tidal data (Fig. 5a). Once again, the 24-h bandsare almost coincident. Moreover, there is remarkable coinci-dence between the other frequency bands obtained from theresiduals and the gravimetric profile (indicated by verticaldashed green lines in Fig. 5a). They are especially pronouncedat ∼21.8 h and 28 h, where the match appears perfect.

Series 2 presents a higher, more abrupt increase in thephoton counts recorded just before and after a New Moon ora Full Moon. These two periods were accompanied by ahigher rate of seedling development, as shown in Fig. 5b.This figure is a 360° polar diagram for both germinationvigor (assessed as the sum of the lengths of coleoptile andfirst root) and the total photon count in relation to the testtime during the lunar phase. The timescale is represented bythe increasingly dark line color for both curves. It is notice-able that the total photon count rises by the same degree asthe rate of seedling growth, with peaks at 20° (just after FullMoon), 125° (middle of Last Quarter), 210° to 230°, and290° to 320° (start of First Quarter) (Fig. 5b). Of note is thelarge variation at the points which coincide with minimalaverage development—i.e., around the days of Last QuarterMoon (40° to 100°) and of New Moon (160° to 190°). Atthese times, seed samples (in triplicate tests) develop well,but many seedlings show weak growth, thus reducing theaverage growth and increasing its variance.

Fig. 4 Series 1. a Selection ofexamples for the photon-countvariation ±δPC (relating to thelocal 24-h linear regression),where there is coincidence be-tween the counts and the gravi-metric tide profile ±δg, and itsfirst time derivative. b The fastFourier transform for the com-plete time course

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The variation of seedling development in relation to lunarphase is often considered a naïve and superstitious assump-tion (Kollerstrom and Staudenmaier 2001). Nevertheless,they were also displayed in long-term experiments byKolisko and Kolisko (1939). Our observations tend to con-firm the Kolisko’s findings.

Discussion

The spontaneous light emitted from germinating wheatgrains which had been placed in a dark chamber was inves-tigated during tens of separate tests. As revealed by linearregression and the residuals obtained therefrom, utilizingseparate 24-h intervals within the entire time course, therewere oscillations in the photon-count curves which appearedto be highly synchronized with the tidal cycle. High andsignificant correlation coefficients occurred not only withrespect to local correlation but also in relation to the overall

correlation between the photon-count inflexion points andthe closest gravimetric tidal extremata. The pattern of theresiduals curve, δPC, when tested by FFT, showed compo-nents very similar to those of the gravimetric tide δg.

The video records obtained in parallel to a series of bio-photon experiments corroborate the proposed relationshipbetween a seedling’s development and its spontaneous lightemission pattern. Because the light emission is associatedwith the presence of O2 radicals, it may be that enzymaticreactions (e.g., via catalases, peroxidases) which release reac-tive oxygen species (ROS) are instrumental in the modulationof this phenomenon, as already demonstrated for oxidativeprocess on human skin (Rastogi and Pospíšil 2011). Thecontinual loading of new cellulosomes (Fontes and Gilbert2010) nanomachines which assist in deconstructing and con-structing plant cell walls and their carbohydrates, across theplasma membrane, may play a part in this. However, wallsynthesis is but one aspect of the present coleoptile andradicular growth system. A second important aspect of thegrowth system is the passage of water into cells in order tomaintain the turgor required for cellular expansion. Again,passage across the cell membrane is a necessity, in this casefor the passage of water. Rhythmic aspects of water passagehave already been discussed in relation to lunisolar gravita-tional force and elongation growth of roots (Barlow andFisahn 2012). A further possibility is that photons are gener-ated in mitochondria and are channeled to the outer regions ofthe cells via microtubules (Rahnama et al. 2011). All thesepossible sources of biophotons in plant cells and tissues arenot mutually exclusive and, hence, the photon counts mayrepresent a composite evaluation of emissions from all thevarious processes by which photons can be derived andrendered observable at the exterior of the seedling.

Conclusion

The results seem to validate the proposed idea that a cosmicclock, which regulates the activities of living beings, isprocessed continuously throughout the solar day. Thisoccurs most evidently in plants, whose biorhythms are pro-posed as being determined by lunisolar gravitational ororbital relations (Klein 2007). We believe such a system tobe regulating the biophoton emission process which wehave observed in the present case.

As presented here, the photon-counting procedureappears to be a fast, non-invasive method to evaluate growthand seedling development in real time. Not only is it a usefultool for tests which involve seedling development and theassociated responses in relation to stressful or toxic substan-ces, but it is also an interesting addition to the methodologyavailable for chronobiological investigations. Furtherexperiments, with similar series of consecutive tests, may

Fig. 5 Series 2. a The fast Fourier transforms for the residuals andgravimetric time curves; b germination vigor (total seedling length)and total photon-count number plotted in polar fashion against thelunar phases; the progress, in time, is indicated by a progressiveintensification of the line colour

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be performed in longer (>2 months) time spans to confirmthe relation of germination performance, biophoton emis-sion, and the moon-phase alternations.

Acknowledgments Authors are grateful to FAPESP (Fundação deAmparo `a Pesquisa do Estado de São Paulo, Brazil) and CNPq(Conselho Nacional de Pesquisa, Brazil) for partial support. C. M.Gallep is particularly thankful to Prof. E. Conforti (FEEC/Unicamp)for his previous support to LaFA group, and to Peter Bieckark (Sítiodas Fontes) for his inspiring thoughts, deeds and discussions.

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