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Keywords:

  • circadian clock;
  • lunar gravity;
  • root elongation kinetics;
  • tidal force;
  • video imaging

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • All living organisms on Earth are continually exposed to diurnal variations in the gravitational tidal force due to the Sun and Moon.
  • Elongation of primary roots of Arabidopsis thaliana seedlings maintained at a constant temperature was monitored for periods of up to 14 d using high temporal- and spatial-resolution video imaging. The time-course of the half-hourly elongation rates exhibited an oscillation which was maintained when the roots were placed in the free-running condition of continuous illumination.
  • Correlation between the root growth kinetics collected from seedlings initially raised under several light protocols but whose roots were subsequently in the free-running condition and the lunisolar tidal profiles enabled us to identify that the latter is the probable exogenous determinant of the rhythmic variation in root elongation rate. Similar observations and correlations using roots of Arabidopsis starch mutants suggest a central function of starch metabolism in the response to the lunisolar tide. The periodicity of the lunisolar tidal signal and the concomitant adjustments in root growth rate indicate that an exogenous timer exists for the modulation of root growth and development.
  • We propose that, in addition to the sensitivity to Earthly 1G gravity, which is inherent to all animals and plants, there is another type of responsiveness which is attuned to the natural diurnal variations of the lunisolar tidal force.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plants grow autotrophically using light, CO2, nutrients and water, all of which they acquire from the abiotic environment. As plants are required to maximize the capture of their resources, they have developed optimized seasonal and daily time-dependent foraging and developmental strategies. Accordingly, plants, like other organisms, have evolved sophisticated biological clocks which are commonly entrained by exogenous environmental cues provided, for example, by photoperiods and by diurnal variations of light spectra, and temperature. All of these, however, occur within the context of the gravitational fields generated by the Earth, Sun and Moon, either singly or in combination.

For many years, the effect of the lunar gravitational force upon the Earth’s gravity field has been discussed in relation to numerous physiological and behavioral processes in plants and animals (Denny & Paine, 1998; Endres & Schad, 2002; Helmuth et al., 2002; Engelmann, 2007; Barlow et al., 2008; Connor & Gracey, 2011; Barlow & Fisahn, 2012). As a result of the rotation of the Earth around its axis, as well as the relative orbital motions of the Earth and its Moon around the Sun, the gravitational field of the Earth is continually modulated by the lunisolar gravitational force (Konopliv et al., 1998, 2001). On Earth, this modulation is most pronounced and readily measurable in relation not only to the tidal movements of the seas and oceans but also to small variable and elastic deformations of the Earth’s crust. These latter are measurable over time by gravimetry (Xu et al., 2004; Crossley et al., 2005) and expressed in variations in Earthly 1G gravity in incremental or decremental units of microGals, where 1G = 9.80 × 108 μGals.

Diurnal variation of the strength of the lunisolar gravitational field underlies the sensation of both duration and time (Dorda, 2010). Generally, estimation of the passage of time is based on cyclical phenomena, as generated by the rotation of the Earth around the Sun, and the causal background for these cyclical phenomena is gravitational forces. Therefore, it is plausible to use gravitation as one explanatory variable (in addition to the daily cycle of day and night) for the perception of time (Dorda, 2010) – a principle understood by marine biologists (Palmer, 1995; Endres & Schad, 2002), the gravitation effect being manifest in the marine tides. In particular, exact determination of the period of a recurring event will enable a distinction to be made between a solar (linked with a 24-h rotational period) and a lunar gravity-associated (24.8-h) zeitgeber, or timekeeper.

Sensing, or perception, of Earthly gravity and the resulting graviresponses are becoming better understood in many plant and animal systems (Hemmersbach & Braun, 2006; Morita, 2010). The vectorial information of 1G gravitational acceleration is a reliable reference for the orientation of growing organs, plant roots in particular (Friml et al., 2002; Moreno-Risueño et al., 2010). Furthermore, the sensing of a periodical alteration of a signal as a result of the lunisolar gravitational force might provide a further, precise exogenous time-keeping mechanism in living organisms (Brown, 1976; Dorda, 2010). The lunisolar gravitational force has usually been overlooked in experimental procedures which have otherwise aimed at being free of external environmental factors of Earthly origin, even though stimuli derived from this force are recognized in many responses of animals to lunar parameters (Zimecki, 2006).

Root extension growth can be measured by using digital calipers to determine root tip displacement, marking root tip position on a transparent surface, or capturing a series of time-lapse images. Commercial software such as Winrhizo (Arsenault et al., 1995), Optimas analysis software (Media Cybernetics, http://www.mediacy.com) and Image J (Abramoff et al., 2004) has been introduced to assess root length and its incremental increase. Recent studies have also quantified differences in root architecture (Armengaud et al., 2009). Using these approaches, it has been shown that there are rapid adaptations of growth in response to modulation of light intensities and photoperiods (Aguirezabal et al., 1994; Muller et al., 1998; Nagel et al., 2006). Moreover, there are different growth zones at the root tip, which are differentially affected by a variety of qualitatively different stimuli (Walter et al., 2003). However, there is little information on how roots respond to diurnal stimuli or to the continuous, but nevertheless naturally occurring modulation resulting from the lunisolar tidal force.

We have developed a system for high-throughput analysis of Arabidopsis thaliana root growth kinetics (Yazdanbakhsh & Fisahn, 2009, 2010). Seedlings are grown on agar plates in a custom-designed controlled climate chamber, in which the roots are illuminated by infrared diodes and are oriented and fixed in the focal plane of a charge-coupled device (CCD) camera which captures changes in the position of a root tip. The images are captured by a time-lapse video system, and root tip positions are determined and an elongation rate estimated. We used this method to investigate the capacity of A. thaliana wild-type seedlings to display robust oscillations in the elongation rate of their primary roots in continuous light. Moreover, we asked if the lunisolar tidal force is the only external geophysical variable that is apparently capable of modulating root elongation rate; and thus if it could function as a zeitgeber under constant light conditions.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant material and growth conditions

Seeds of Arabidopsis thaliana (L.) Heynh wild-type Columbia (Col-0) and sex1 mutants (Caspar et al., 1991) were surface-sterilized for 20 min with 10% sodium hypochlorite solution containing 0.1% surfactant (Triton X-100), rinsed several times with sterile water and plated on the surface of solid nutrient agar (7.0% m/v; SELECT agar; Invitrogen, Karlsruhe, Germany) supplemented with half-strength Murashige–Skoog medium (Murashige & Skoog, 1962; M02 555, pH 5.6; Duchefa, Haarlem, the Netherlands). After 4 d of stratification, the seeds were placed in Petri dishes oriented vertically in a phytotron (constant day and night temperature of 21°C ± 0.5°C [correction added after online publication 29 May 2012: in the preceding text, the previously published temperature 210°C has been corrected to read as 21°C]; 100 μmol m² s¹ photon flux density (5x Philips TL-D18.830 and 5x Philips TL-D 18.840 fluorescent light tubes; Growland, Hamburg, Germany). At day 9, seedlings that had developed roots of > 1 cm length were selected and distributed evenly in a row 3 cm from the top of a vertically oriented surface of nutrient agar in 120 × 120 mm rectangular Petri dishes, with 15–25 seedlings per plate. After 2 further days of acclimation in the phytotron, Petri dishes containing the seedlings were used for root length measurement.

Image acquisition and root elongation analysis

Root growth kinetics were collected as described previously (Yazdanbakhsh & Fisahn, 2009, 2010). In brief, a custom-designed phytochamber housed the central measuring head of the plant root monitor (PlaRoM; Yazdanbakhsh & Fisahn, 2009). The actinic photon flux density in the chamber at the surface of the leaves of the seedlings was 90 μmol m−2 s−1. Temperature was controlled by a cooling device providing ± 0.5°C accuracy. The PlaRoM imaging platform screens the surface of two Petri dishes simultaneously and captures time-lapse records of the seedlings growing upon them (Yazdanbakhsh & Fisahn, 2009). Image stacks were collected by a CCD camera (Panasonic Colour CCTV Camera, WV-CP210/G; Matsushita Communication Industrial Co. Ltd, Tokyo, Japan) mounted on the video port of the microscope. To monitor the seedlings regardless of their actinic light requirements, an infrared light source (Infra-Red Illuminator CE-7710; Jenn Huey Enterprise Co., Ltd, Taipei, China) provided illumination. Screening of the Petri dish and the capture of time-lapse records was controlled by the PlaRoM imaging software application (Yazdanbakhsh & Fisahn, 2010). The magnification of the microscopes was set so that the video stream covered a 4.58 × 3.33 mm area of the surface of the Petri dish. This allowed images to be captured with a resolution of 5.96 μm × 5.78 μm per pixel. The root extension profiling software analysed the time-lapse records and provided the growth-velocity profiles (Yazdanbakhsh & Fisahn, 2010).

Lunar gravity profiling

A program (Etide) was used to calculate the gravimetric tide which stands proxy for lunisolar tidal force. Etide is based upon the 50 parameters used by Longman (1959) for computing the vertical gravimetric component of the lunisolar tidal force, the horizontal component of which is found to be negligible. Using an elasticity factor of 1.16 for the body of the Earth, the Etide program estimates the tidal rise and fall of the Earth’s gravitational acceleration at any given location brought about by the combined gravitational action of the Sun and Moon – hence the term lunisolar tidal acceleration. About 30% of this acceleration is attributable to the mass of the Sun and c. 60% is attributable to that of the Moon. The input to Etide consists of the latitude, longitude and altitude of the location in question (Potsdam; 54°24′N, 12°58′E), together with the calendar dates for which estimates are required. The output consists of gravimetric values in microGals (the gravitational acceleration at the Earth’s surface; 1G = 9.8 × 108 microGals; 1Gal = 1G = 9.80665 m s−2), estimated at 15-min intervals over the required period. The microGal time-courses are prepared with reference to UTC (GMT). They were then adjusted to the local times at which the time-courses of the biological data had been prepared. The original program of Longman (1959) was checked against earlier computations and found to provide similar estimates of tidal acceleration in microGals. The output from the present Etide program was also checked against values from a similar, but independent, program provided by Professor J. Střeštík (Institute of Geophysics, Academy of Sciences, Prague, Czech Republic).

Statistical methods

Cross-correlation methods were used according to the Wessa program available on the Internet (Wessa, 2012). Local tracking correlation was performed using the methodology of Papadimitriou et al. (2006).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The growth of A. thaliana seedling roots at a constant temperature of 21°C was entrained by either 12-h (Fig. 1, red trace,) or 16-h (Fig. 1, blue trace) photoperiods (12 h : 12 h or 16 h : 8 h, light : dark). Under these conditions, which were applied for 4 d, the kinetics of root elongation exhibited four main features: (1) an increased elongation rate during the dark period, and (2) a brief acceleration of elongation shortly after the commencement of the light period, followed by (3) a decline of the elongation rate during the major part of the light period with an acceleration of root elongation near the end of the light period (Yazdanbakhsh & Fisahn, 2011). The accelerated elongation noted at (1) and (2) may be attributable to some feature of water and/or carbon utilization by the seedling in the respective light–dark regime, these two factors being likely driving forces for root growth. A fourth feature, which only appeared following the entrainment period, when the roots were placed in continuous illumination, was a persistent diurnal oscillation of elongation rate (Fig. 1) (see also Fig. 1, Yazdanbakhsh & Fisahn, 2011; and see Fernandez & Wagner, 1994 for analogous observations on stem growth). The source of this oscillation is unknown, but the fact that it continues in constant illumination, when the elongation rate is increasing, does not seem to favour an endogenous, physiological origin, but suggests that an exogenous mechanism may be operating. Particularly interesting in this respect is that the period of oscillation was c. 25 h, close to that of the lunar day of 24.8 h. This congruence justifies examination of a possible participation of the lunisolar gravitational force in modulating root growth under the present free-running conditions following transfer of the roots into continuous low-intensity light from their 16 h : 8 h (light : dark) entrainment regime.

image

Figure 1. Averaged kinetics of root elongation obtained during the transition from diurnal to continuous illumination. Blue line: roots of 11-d-old Arabidopsis thaliana seedlings were entrained using photoperiods of 16 h : 8 h, light : dark (n = 23). Sharp transient peaks in root growth rate were induced by the onset of light. Black symbols on the root growth characteristic at days 1–3 denote the dark period. At day 3, the light protocol was changed so that the seedlings were exposed to continuous illumination. Under these conditions, root growth rate exhibited a slow oscillation that was offset by a logarithmic drift. Red line: roots of 11-d-old A. thaliana seedlings were entrained using photoperiods of 12 h : 12 h, light : dark (n = 9). Then, on day 19, root extension rates were measured during two 12 h : 12 h cycles. Black symbols on the red trace on days 1–3 denote the dark period. Sharp transient peaks in root growth rate were induced by the onset of light. Subsequently these roots were exposed to 5 d of continuous illumination. Under these conditions root growth rate exhibited a slow oscillation that was offset by a logarithmic drift. Plots are of the mean elongation rates (μm min−1). Error bars denote SE.

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Under alternating light–dark conditions (16 h : 8 h) (Fig. 1), the profile of root elongation recorded was apparently defined by the photoperiod (see Fig. 1 of Yazdanbakhsh & Fisahn, 2011) However, placing these same roots under conditions of continuous light also led to a slow oscillation of elongation rate, such as described above, the mediators of which are not obvious. Previous studies by Barlow et al. (2010) and Barlow & Fisahn (2012) have indicated that oscillatory leaf movements of bean (Canavalia Ensiformis) plants are regulated by the passage of the Moon and the attendant changes in the lunisolar tidal force. Therefore, in order to analyse the putative involvement of a lunisolar gravitational component in the modulation of root growth kinetics, as revealed by the oscillations of the root elongation rates, we compared high-resolution profiles of root growth rate with the estimated contemporaneous lunisolar tidal force for the geographical location where the experiments were performed (Fig. 2). These latter profiles consist of estimates from the Etide program of the ‘gravimetric tide’, a measure of the wave of deformation passing across the Earth’s crust at a given location. Comparison between the root growth rate and the lunisolar tidal force revealed a consistent congruence between the daily minimum in root growth rate and a corresponding dominant minimum in the lunisolar gravity profile (Fig. 2a). Local cross-correlation tracking (Fig. 2b) confirmed the visual alignment of root growth rate and lunisolar tidal force, with the highest correlation coefficient coinciding with the respective minima in the two time-courses. Final conclusive evidence for a near 25-h periodicity of the root growth rate emerged from the alignment of root growth rate data with a 24-h and a 24.8-h sine wave (Fig. 2c,d). Cross-correlation substantiated the inherent 24.8-h periodicity (the period of the lunar day) of the root growth rate (Fig. 2e,f).

image

Figure 2. Comparison between root elongation rates (blue lines) after transfer from a photoperiod of 16 h : 8 h, light : dark to continuous illumination, as well as the lunisolar tidal profiles (green line) and a 24.8-h (24-h) sine wave (green line). Roots of Arabidopsis thaliana seedlings were entrained using photoperiods of 16 h : 8 h, light : dark (n = 23). At day 15, the light protocol was changed so that the seedlings were exposed to continuous illumination. All growth rate kinetics depicted refer to times in continuous illumination. (a) The blue trace denotes the average in root elongation rate (μm h−1) of seedlings (n = 23) in continuous illumination. The green trace indicates the lunisolar gravity profile (in μGal). Vertical lines specify the positions of the joint troughs in the root growth rate and the lunisolar gravity profile. NA, normalized amplitude of root growth rate. (b) Local correlation score according to Papadimitriou et al. (2006) of the two data profiles (averaged root growth rate and lunisolar gravity profile) depicted in (a). Black arrows indicate sites of high local cross-correlation scores between the respective troughs in root growth rate kinetics and lunisolar tidal profiles. LCS, local correlation score. (c, d) Alignment of averaged root growth rate profiles (after subtraction of a logarithmic drift; blue lines) with a 24.8- and 24-h sine wave (green traces). (c) Averaged growth rates (blue line) of n = 23 seedlings that were exposed to continuous illumination were aligned with a sine wave of 24.8-h periodicity (green line). Before alignment, a drift elimination was performed to compensate for the gradual increase in root growth rate caused by continuous illumination. (d) Averaged growth rates of n = 23 seedlings (blue line) that were exposed to continuous illumination were aligned with a sine wave of 24-h periodicity (green line). Drift elimination was performed on the root growth rate profiles as mentioned in (c). Visual inspection of the two alignments presented in (c) and (d) clearly indicates that the periodicity inherent to root growth is 24.8 h, the exact period of the lunar day. (e) Cross-correlation function (CCF) of the alignment presented in (c) (24.8 h): CCFmax = 0.853 with k = 0. (f) Cross-correlation function of the alignment presented in (d) (24 h): CCFmax = 0.783 with k = 4. Dotted blue lines indicate the upper and lower confidence bounds (Wessa, 2012). This statistical analysis confirms that the periodicity immanent to the changes in root growth is equivalent to the lunar day, 24.8 h.

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After transfer of the roots from a 12 h : 12 h photoperiod to continuous illumination, time-courses of their elongation rates were compared with those defining the lunisolar tidal profiles (Fig. 3a). An exponential drift was applied to the lunisolar tidal force profile and, for clarity, we have superimposed this drift upon the similar natural drift displayed by the profile of the root growth rates (Fig. 3a). The latter drift is initiated when the seedlings are first exposed to continuous light. It should be noted, however, that the drift in the root growth data is not necessarily perfectly logarithmic. In keeping with many time-courses of plant growth, the relationship is probably logistic, and may correspond with the post-germination establishment. Further analysis of the mode of drift of the root elongation rate could further improve the correlation coefficients between the biological and geophysical profiles.

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Figure 3. Comparison between root elongation rates in Arabidopsis thaliana (blue line) after transfer from a 12 h : 12 h, light : dark photoperiod to continuous illumination, as well as the lunisolar tidal profiles (green line). (a) Alignment of averaged root elongation rates (blue line) and the corresponding lunisolar gravity profile (green line). An exponential drift was superimposed upon the lunisolar gravity profile (green line) to match a similar exponential drift in root elongation rates encountered when seedlings (n = 9) were exposed to continuous illumination (blue line). The green peaks at the bottom of the graph indicate joint segments of the graphs of both root elongation rate and lunisolar gravity where there were turning points (i.e. slopes of low or zero value). (b) The cross-correlation functions (CCFs) estimated in selected segments of the graphs of root elongation rates and lunisolar gravity data depicted in (a).

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We identified segments of each time-course, biological and geophysical, which exhibited a common joint trend. Of special interest were those segments that were characterized by a small, or zero, slope (i.e. the turning points in the graphical time-courses or profiles). Green peaks in Fig. 3(a) indicate these areas. As seen in Fig. 3(a) most of these points in the gravity profile are mirrored in the root growth profile, where they coincide with portions of the growth curve having small, or zero, slope. This coincidence strongly suggests the lunisolar gravity pattern to be a possible modulator of root growth.

Cross-correlation analysis of selected portions (those encompassing the turning points in both data sets) of the time-courses relating to root elongation rate and the lunisolar tidal force provided a correlation coefficient of r = 0.90 (Fig. 3b).

To investigate whether the slow oscillation in root elongation rate resumed following a cessation of root growth, another set of A. thaliana seedlings were exposed to continuous illumination after a period of 4 d in continuous darkness which inhibited growth (i.e. there was no entrainment period of alternating light and dark). The elongation rate recovered slowly, increasing during the first 36 h in the illuminated environment, and then continuing to accelerate. A slow oscillation was apparent in the profile of root elongation rate (Fig. 4a). The temporal pattern of the oscillation was in perfect correspondence with the rise and fall of the gravimetric tide. Cross-correlation between segments of the time-courses of root elongation rates and the lunisolar tide showed correlation coefficients of r ≥ 0.95 (Fig. 4b). Moreover, cross-correlation revealed a delay of k = 0 h, thereby uniquely associating the oscillation of the root elongation rate with the time-course of the changing lunisolar tidal force: that is, the tested segments of the two time-courses were synchronous.

image

Figure 4. Alignment and cross-correlation of root elongation rates and lunisolar gravity profiles. (a) Seedlings of Arabidopsis thaliana (n = 6) were entrained in a 16 h : 8 h, light : dark photoperiod for 20 d. The seedlings were then exposed to continuous darkness for 4 d before being continuously illuminated. After 2 d in continuous light, root growth kinetics (blue line) matched the lunisolar gravity profile (green line). (b) The cross-correlation function (CCF) between root growth rate and lunisolar gravity profile depicted in (a) yielded a maximal r = 0.954, and a lag k = 0 h. (c) Seedlings of A. thaliana Col-0 (n = 11) were entrained in a 12 h : 12 h, light : dark photoperiod for 16 d. Seedlings were dark-grown for a further 4 d to reduce the root growth rate. Recovery of root growth in continuous light occurred after a delay of almost 36 h. Final recovery of root elongation (blue line) was accompanied by an oscillation of elongation rate that coincided with the lunisolar tidal profile (green line). (d) Cross-correlation between the root elongation rate and the lunisolar tidal force depicted in (c) provided a maximal value (CCFmax) of r = 0.96 and a lag k = 0 h. (e) Seedlings of the A. thaliana sex1 mutant (Caspar et al., 1991) (red line; n = 5) experienced the same light protocol (c) as Col-0 wild type. Recovery of root growth rates after 4 d of darkness occurred with slightly modified kinetics as compared with the Col-0 wild type. Alignment of the root growth rate profile with the corresponding lunisolar tidal profile (green line) provides a good visual concordance. (f) Cross-correlation between the root elongation rate and the lunisolar tidal force provided a maximal value of (CCFmax) r = 0.9 and a lag k = 0 h, as depicted in (e).

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To substantiate that the rhythmicity of root elongation rates in free-running conditions paralleled the inherent variations in lunisolar gravity, we compared the above-mentioned results, which were obtained in the winter of 2008, with those of experiments performed in the summer of 2007, using seedlings entrained to a 12 h : 12 h photoperiod (Fig. 4c). Similar to the experiments performed in the winter of 2008 (with seedlings that had been entrained to 16 h : 8 h photoperiods (Fig. 4a)), a strong correlation emerged between the two variables (Fig. 4c). In each case, the turning points in root elongation rate were synchronized with turning points in the lunisolar gravity profile. Cross-correlation performed with data pertaining to each set of seedlings provided coefficients of r = 0.96 and a delay k = 0 h (Fig. 4d). Thus, under these two contrasting seasonal conditions of summer and winter, there was a demonstrable independence of the exogenous origin of the slow oscillation in root elongation rate, a common factor in each case being the rhythm of the lunisolar tidal acceleration.

When the process of root growth has been examined in relation to Earthly gravity (i.e. the graviresponse following experimental displacement of a root in the gravity field), the extent to which growth is affected has been found to correlate with the starch content of the root apex (Caspar et al., 1991; Yu et al., 2001; Morita, 2010). Therefore, to investigate the effects of modulation of starch metabolism on rates of root elongation and its oscillating pattern, and also their relationships to the lunisolar gravity profile, we exposed the A. thaliana starch excess mutant, sex1 (Caspar et al., 1991; Yu et al., 2001), to the previously mentioned light protocol of extended darkness and subsequent continuous illumination (Fig. 4e). The sex1 mutant lacks a glucan water dikinase (GWD), which is required to phosphorylate starch and, hence, these plants are impaired in starch degradation. The mutant contains large amounts of starch within the chloroplasts of the shoot tissue, which is broken down slowly during the night (Caspar et al., 1991; Yu et al., 2001). As indicated by cross-correlation, a positive relationship exists between the oscillations of mutant root growth rate and the pattern of the lunisolar tidal force. However, the correlation was not as pronounced in the mutant as it was in the case of the Col-0 wild-type plants. Fine-tuning of the root growth rate in the sex-1 mutant in accordance with the lunisolar gravity profile was apparently lacking at the turning points in the gravity curve (Fig. 4f).

Previous investigations of the lunisolar response mechanism in animals (Wikelski & Hau, 1995) and bean plants (Barlow et al., 2008) found a unique phase shifting capacity coincident with the response to gravity. To demonstrate a similar phase shift in elongating roots of A. thaliana, we studied the effect of light-to-dark transitions on rhythmic root growth. Hence, a further set of A. thaliana Col-0 seedlings were grown under continuous illumination, from the seed imbibition stage onwards (i.e. there was no light/dark entrainment period). Root growth of 14-d-old seedlings was recorded over a period of 102 h (Fig. 5a,b) and the elongation rates estimated at the customary 0.5-h intervals. The root elongation rates of these seedlings which had never been exposed to darkness (cf. those rates obtained following alternating light/dark periods) oscillated in the same manner as already described. However, the rhythmic elongation pattern for the individual roots did not show a robust synchrony. Two simultaneously recorded individual root growth profiles are shown in Fig. 5(a,b). When compared with the lunisolar tidal force profile, it is apparent, nevertheless, that the growth rhythm of each root matches certain features of the tidal force profile (Fig. 5a,b): each individual growth rate time-course exhibits maxima or minima that coincide with one or other extremata of the tidal force profile. However, because the roots probably developed at different rates and commenced their putative lunisolar gravity-modulated oscillation at different times during their post-germination development, a complete synchrony is lacking between two of the observed root growth profiles. Hence, at the commencement of their growth, it was not predictable which of the forthcoming turning points would be responded to by any given root. It may be that some entrainment period (either of alternating light/dark or a continuous dark period before continuous light) is a necessary precondition for the establishment of a starting point for the lunisolar-driven oscillation.

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Figure 5. Comparison of root elongation kinetics obtained from seedlings raised in continuous light (never exposed to darkness) together with the corresponding lunisolar gravity profiles. (a) Oscillations of root elongation rate (blue line) in a seedling of Arabidopsis thaliana raised in continuous light follow the oscillations of the lunisolar gravity profile (green line). A decrease in elongation rate always coincided with a local maximum (peak) in the lunisolar gravity profile. (b) Growth rates of a second seedling (blue line) pretreated in identical conditions as in (a) were recorded simultaneously to that depicted in (a). As time passes, the peak of maximum elongation rate switches from one peak in the gravity profile (green line) to the next. Initially, root growth rate decreases whenever a local minimum occurs in the lunisolar gravity profile. At day 3, a decline in root elongation rate coincides with a local maximum in the lunisolar gravity profile. (c) Alignment of the average of three roots (the growth rate profile of the third seedling is not depicted as it closely resembled that of seedling 1) of seedlings (blue trace) that were never exposed to darkness with a 24.8-h sine wave (green trace). (d) Alignment of the average of three roots of seedlings (blue trace) that were never exposed to darkness with a 24-h sine wave (green trace). (e) Alignment of the average of three roots of seedlings (blue trace) that were never exposed to darkness with the lunisolar tidal profile (green trace). (f–h) Cross-correlation functions of the data sets shown in Fig. 5(c–e). (f) Cross-correlation function between the average of three roots of seedlings that were never exposed to darkness and a 24.8-h sine wave (data according to Fig. 5c). Cross-correlation performed on these two time series exhibited a CCFmax = 0.883 (Supporting Information Table S1). (g) Cross-correlation function between the average of three roots of seedlings that were never exposed to darkness and a 24-h sine wave (data according to Fig. 5d). Cross-correlation performed on these two time series exhibited a CCFmax = 0.81 (Table S1). (h) Cross-correlation function between the average of three roots of seedlings that were never exposed to darkness and the lunisolar tidal profile (data according to Fig. 5e). Cross-correlation performed on these two time series exhibited a CCFmax = 0.71 (Table S1).

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It should be noted that, in Fig. 5(a,b), the lunisolar gravity profile is characterized by pairs of maxima and minima (peaks and troughs) each day, whereas in the previously shown profiles (Figs 3a, 4a) only a single peak or trough daily is evident. Each type of pattern is characteristic of different days during the lunar month, semidiurnal, equal tides being characteristic of new Moon and full Moon, whereas diurnal tides and semidiurnal equal tides are characteristic of first- and last-quarter Moons. Statistical analysis performed on the averages of three seedling roots never exposed to darkness confirmed the 24.8-h periodicity in the root growth rate kinetics (Fig. 5c–e) and revealed a high score in cross-correlation between biological and geophysical data (Fig. 5f–h).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Comparison of root growth kinetics and lunisolar tidal profiles revealed high degrees of synchrony not only between these two biological and geophysical variables but also between the individual roots in any given experiment (Figs 1–5). It is important to note that, in the present analysis, we focus on the time-courses for the free-running situation, where there is no input into the biological system from potentially variable environmental factors inherent to the growth cabinet, such as temperature and light/dark periods. It is, however, impossible to limit the influence of all external geophysical variables, such as lunisolar tidal force and atmospheric electric fields, upon the biological material, because these variables are ever-present in the external environment. The lunisolar tidal force does, however, vary in amplitude and phase during the course of each day; and it is this variation that allows the possibility of examining whether the time-courses for the biological material vary similarly. Hence, congruence in phase and periodicity between the lunisolar and the biological profiles uniquely suggests the exogenous origin of the oscillation in root growth rate. Although lunisolar gravitational acceleration is an obvious candidate for consideration in relation to variation in the time-course of change in the biological system, it should not be regarded as the only possibility. A holistic view of exogenous influences would also include variations in atmospheric electricity and barometric pressure, as well as various types of radiation, and geomagnetic effects, all of which themselves can be subject to lunar modulation (Markson, 1971; Mehra, 1989). Nevertheless, lunisolar tidal force is the most persuasive candidate, not least because the cross-correlations we present, and especially the time-delay parameters, k, suggest an immediate and continual interaction between the tidal force and the root elongation process.

Previous studies demonstrated that, in comparison to the elongation of A. thaliana roots, the unforced, or free-running, leaf movements of bean plants might be regulated by the lunisolar tidal force (Barlow et al., 2008; Barlow & Fisahn, 2012). Earlier experiments had been performed by both Brouwer (1926) and Kleinhoonte (1929, 1932), who attempted to regulate the period of the leaf-movement rhythms and thereby to discover the mechanisms of the putative interaction between the plant and its environment. Both workers used a growth chamber where they could experimentally alter the duration of the light and dark periods while keeping constant the temperature and humidity. Experimental lighting regimes, such as 18 h : 6 h, 16 h : 16 h, 12 h : 12 h (light : dark), and so on, certainly produced alterations in the period of leaf movements. However, because the turning points in the profile of δg themselves occur c. 6–8 h apart (i.e. times that were multiples of the light and dark durations), the new leaf turning points entrained by these new light/dark conditions often continued to coincide with the δg turning points (Barlow & Fisahn, 2012). Hence, it is probable that the lunisolar geophysical signal which affects the bean leaf movement also affects the oscillations in root elongation growth of A. thaliana.

Leaf movements are not necessarily synchronous among the different plants in a single experimental group of newly germinated seedlings (Alford & Tibbitts, 1970; Klein, 2007). A similar asynchrony was observed in the growth rate profiles of roots that were never exposed to darkness (Fig. 5a,b). Moreover, we demonstrated that single turning points in the lunisolar gravity profile can be ignored by the root response when the lunar gravity profiles exhibit two maxima or minima within 24.8 h. In the presence of two clocks with unequal periodicities of 24 and 24.8 h (Connor & Gracey, 2011), a favourable synchrony between extremata of these two time keepers will progressively deviate daily. To reset the beneficial synchrony of the two clocks, a phase-shifting mechanism that enables to skip the response to one or other extremata in the lunisolar tidal profile could provide a developmental advantage in optimization of foraging strategies (Wikelski & Hau, 1995). Thus, under appropriate environmental conditions and specific characteristics in the lunisolar tidal profile, Fig. 5(a,b) indicates that roots of A. thaliana are capable of such a beneficial phase adjustment between the progressively (daily) deviating phase relations of the circadian and the lunisolar tidal clocks.

An apparent concordance between the variation in the gravimetric tide, δg, and the daily variation in stem diameter, δD, was demonstrated by Zürcher et al. (1998). Two evergreen softwood trees of Norway spruce (Picea abies) were placed in darkness within a constant environment chamber. Under such circumstances, transpiration of the trees was much diminished and showed no fluctuation which, had it occurred, might have accounted for the diurnal variation of δD. Apart from a concurrent variation in geomagnetic flux, as measured by the Polar Cap Index (Barlow et al., 2010), the only known variable that might significantly have affected δD was δg, the variation in the gravimetric tide. Unlike the already described leaf movements, which oscillate sharply between two defined limiting positions, ‘down’ and ‘up’, the variations of δD show a sinusoidal wave-form. This being so, it is quite straightforward to perform cosinor cross-correlation analysis, by which means it was established that, over the 3 d of observation, a significant relationship existed between δD and δg with a time delay, k, of c. 1 h (Barlow & Fisahn, 2012). Additional analyses of original data on δD, obtained by Cantiani et al. (1994), similarly confirmed a relationship between δD and δg. The tree species studied included three other evergreen gymnosperms and four deciduous hardwood species, Juglans regia, Liriodendron tulipifera, Mespilus germanica, and Tilia cordata (Barlow et al., 2010). While one should be cautious that quantifiable environmental variations, such as relative humidity and temperature, might influence the range of the variation δD, or interfere with the accuracy of its measurement (see Innamorati et al., 1980), the correspondence between the lunisolar gravity profile and the variations in tree stem diameter for all the mentioned species was highly significant (Barlow et al., 2010).

The intertidal zone of wave-swept sandy and rocky shores has become a model system with which to study the interaction of lunar gravity-induced tidal effects on plants and animals (Denny & Paine, 1998). As the tides rise and fall, areas on the shore are alternately immersed and exposed. The concomitant change from marine to terrestrial conditions can place extreme demands on the physiology of intertidal organisms. To match these demands, intertidal inhabitants evolved biological clocks which are claimed to be linked to the marine tidal cycle and superimposed upon the photoperiod (Connor & Gracey, 2011). The mechanism of this interaction between the 24-h (photoperiod, solar day) and the 24.8-h (tidal rhythm, lunar day) periods in terms of gene expression patterns has not been understood to date (Connor & Gracey, 2011). As demonstrated by our root elongation kinetics (Figs 1–5), plant roots are also capable of adjusting their root growth patterns from a 24-h periodicity under diurnal light/dark conditions to a lunar 24.8-h periodicity when exposed to constant environmental conditions. Moreover, a number of intertidal organisms, particularly crustaceans, display entrained behavioural rhythms that follow persistent circadian (Reid & Naylor, 1989) and tidal patterns (Cohen et al., 2010; Wilcockson et al., 2011), even when held under constant conditions. These findings have led to questions regarding the existence of a tidal clock and the nature of its regulation. Our results (Figs 1–5) are consistent with the existence of a lunar tidal rhythm in elongating roots of A. thaliana under constant, free-running conditions, and thus provide an experimental system with which to reveal potential interaction networks between the lunisolar tidal clock and the day-and-night circadian clock.

The particular free-running conditions used, of continuous low light intensity, may have some bearing on the expression of the lunisolar-regulated oscillation of root elongation. For example, in observations on both crabs (Barnwell, 1966) and a single rat (Brown et al., 1956), the respective activity patterns appeared to be temporally regulated according to the lunar day when the organisms were placed in low-light conditions, but this tracking was lost, and a more constant circadian rhythm followed, when the organisms were placed in conditions of alternating light and darkness.

Recently, a model has been described which provides a quantum gravitational hypothesis for the putative mode of interaction between the lunar gravitational field and a gravity-dependent coherent state of a cluster-like molecular unit located on the surface of the Earth (Dorda, 2004, 2010). In biological materials, this coherent molecular unit can be associated with intracellular water molecules; such a coherent unit could be influential in the growth of a cell. Coherent units of particle assemblies are generally described by quantum electrodynamical equations (Von Klitzing et al., 1980; Del Giudice & Preparata, 1994). They are characterized by their size, the total number of particles in the unit, and their unique dynamic behaviour, as enforced by surrounding electromagnetic or gravitational fields. In mathematical terms, the coherent state is characterized by the interrelation of the de Broglie wave of the molecular unit and a derived orbital time unit of lunar movement (Dorda, 2010). By analogy to the quantum Hall effect, which is related to the macroscopic coherent state of electrons (Von Klitzing et al., 1980; Prange & Girvin, 1987), we may assume that new collective states which result from an effect of lunar motion are characterized in a similar manner by the coherence between mass particles of water (Del Giudice & Preparata, 1994; Arani et al., 1995). It is the interaction of this lunar gravitational field with a defined number or mass of water molecules that induces the coherent state of water molecules. A significant consequence of the interrelation between the quantized lunar gravitational field and the coherent state is the conversion of lunisolar gravitational dynamics, expressed by δg, into the temporal change of the number of coherent water molecules within cells. Thus, maintenance of the coherent state requires increases or decreases in the number of water molecules within the cells whenever the lunar gravitational quantum number is modulated as a result of the rotation of the Earth around its axis. Therefore, the quantized lunar gravitational field provides a timer with a periodicity of 24.8 h, matching exactly the timing of the described oscillations of root elongation as a result of variations in the coherent state of cellular water (Figs 1–5).

On the basis of the described results, we suggest that the extremata in the lunisolar tidal force profile function as exogenous oscillatory timing devices for the roots of A. thaliana. This device comes into play whenever light/dark or temperature entrainment signals are weak or absent. This oscillator could be a result of the relative motions of Earth and Moon during their passage around the Sun, which thereby provides a back-up clock for the modulation of root elongation. It is also possible that whenever a daily solar entrainment for growth is absent, as in the case of roots grown in either continuous artificial illumination or continuous darkness (see the observations of Head (1965) and of Iijima et al. (1998)), the response of the root to the ever-present lunisolar tidal force is rendered apparent. It is in this sense that the tidal force provides a chronometric back-up for the entrainment of root growth.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by the Max Planck Society and by a fellowship to N.Y. We also thank Professor Daniel Robert (Bristol University, UK) and Professor Ernst Zürcher (Bern University, Switzerland) for their valuable support. Prof. Dr. Gerhard Dorda (University of the Armed Forces, Munich, Germany) is gratefully acknowledged for reading the manuscript and providing valuable comments.

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Table S1 Scores of cross-correlations performed with a sine wave of either a 24.8-h or a 24-h periodicity and recorded root growth kinetics under various light entrainments

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