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Advances in Space Research 39 (2007) 1210–1217

Cyclotron-based effects on plant gravitropism

E. Kordyum a,*, M. Sobol a, Ia. Kalinina a, N. Bogatina b, A. Kondrachuk c

a Institute of Botany, National Academy of Sciences of Ukraine, Tereschenkivska St., 2, 01004 Kyiv, Ukraineb Institute for Low Temperature Physics & Engineering, National Academy of Sciences of Ukraine, Lenina Av., 47, 61100 Kharkiv, Ukraine

c Institute of Physics, National Academy of Sciences of Ukraine, Nauki Av., 46, 03650 Kyiv, Ukraine

Received 30 October 2006; received in revised form 23 March 2007; accepted 28 March 2007

Abstract

Primary roots exhibit positive gravitropism and grow in the direction of the gravitational vector, while shoots respond negatively andgrow opposite to the gravitational vector. We first demonstrated that the use of a weak combined magnetic field (CMF), which is com-prised of a permanent magnetic field and an alternating magnetic field with the frequency resonance of the cyclotron frequency of cal-cium ions, can change root gravitropism from a positive direction to negative direction. Two-day-old cress seedlings were gravistimulatedin a chamber that was placed into a l-metal shield where this CMF was created. Using this ‘‘new model’’ of a root gravitropic response,we have studied some of its components including the movement of amyloplasts-statoliths in root cap statocytes and the distribution ofCa2+ ions in the distal elongation zone during gravistimulation. Unlike results from the control, amyloplasts did not sediment in thedistal part of a statocyte, and more Ca2+ accumulation was observed in the upper side of a gravistimulated root for seedlings treatedwith the CMF. For plants treated with the CMF, it appears that a root gravitropic reaction occurs by a normal physiological processresulting in root bending although in the opposite direction. These results support the hypothesis that both the amyloplasts in the rootcap statocytes and calcium are important signaling components in plant gravitropism.� 2007 COSPAR. Published by Elsevier Ltd. All rights reserved.

Keywords: Combined magnetic field; Calcium; Cress; Gravistimulation; Gravitropism; Root cap

1. Introduction

One of the main areas of research in plant biology isrelated to understanding the mechanisms of plantresponses to environmental stimuli because these responsesare critical for plant growth and development. Despite this,the physiological mechanisms by which plants respond tochanges in their environment, particularly in gravisensingand graviresponses, are not clear. For example, althoughsome data suggest that Ca2+ and cytoskeleton participatein graviperception and signal transduction (Lee et al.,1983; Hepler and Wayne, 1985; Moore, 1985; Roux,1990; Wendt and Sievers, 1989; Busch et al., 1993; Sinclairand Trewavas, 1997; Trewavas and Malho, 1998; Knight,

0273-1177/$30 � 2007 COSPAR. Published by Elsevier Ltd. All rights reserv

doi:10.1016/j.asr.2007.03.084

* Corresponding author.E-mail address: cell@svitonline.com (E. Kordyum).

2000; Kordyum, 2003; White and Broadley, 2003; Braunand Limbach, 2006), evidence for Ca2+ participation inthese processes is lacking. Therefore, new experimentalapproaches are required to elucidate the roles of calciumin the gravitropic response.

In 1985, Blackman and Liboff independently suggestedthat the resonance magnetic effects in biosystems involvea specific type of resonance, cyclotron resonance. One ofthe developed models of these effects is parametric reso-nance model proposed by Lednev (1991, 1996). Its basicelement is a degenerate three-dimensional charged oscilla-tor physically realized by a bare ion located in a proteincomplex like calmodulin. We were the first to report (Kord-yum et al., 2005) that CMF with the cyclotron resonancefrequency of calcium ions affects root gravitropism. Resultsfrom this study showed that gravistimulation of cress rootsin this field changed a positive gravitropic response of theroot to a negative response. In the present study, we

ed.

E. Kordyum et al. / Advances in Space Research 39 (2007) 1210–1217 1211

continue these investigations on root gravitropic reactionsin the CMF using light and confocal laser microscopy.We have analyzed the distribution of Ca2+ ions in the rootdistal elongation zone after gravistimulation in the pres-ence of CMF. The results from these studies are relatedto theories on the effects of cyclotron resonance on biosys-tems (Blackman et al., 1985; Liboff, 1985) and ion paramet-ric resonance models (Lednev, 1991, 1996).

2. Materials and methods

The experimental setup with l-metal shield wasdescribed previously (Kordyum et al., 2005). The combinedmagnetic field consisted of permanent magnetic field (BDC)and alternating low frequency magnetic field (BAC) collin-ear to BDC (the magnitudes BAC and BDC were �50 lT),these were measured and controlled by sensor elements(flux-gate magnetometer and SQUID). The control sam-ples were placed into the same position of the shielded vol-ume with a static magnetic field (SMF). The amplitude ofthe wave component was less than 0.05 lT.

2.1. Plant material

Two-day-old cress (Lepidium sativum L.) seedlings wereused in these investigations. Straight, vertically orientedroots from plants that were grown in pure distilled wateror supplemented with 0.44 g/l CaCl2 were gravistimulatedin a moist chamber and placed into a shielded chamberwhere the combined magnetic field (CMF) was created.Experiments were performed in darkness at 21 ± 1 �C.

The angle of curvature of roots from their initial hori-zontal position was measured in 10 min intervals for60 min. The angles were measured from digital imagesusing computer-based image analysis. The experimentswere repeated three times for each condition. Dataare shown as the mean ± SE, and statistically tested usingt-test.

2.2. Light microscopy

Root apices (5 mm long) were fixed in 1% (v/v) glutaral-dehyde and 5% (v/v) formaldehyde (1:1) on cacodylate buf-fer (pH 7.2) after 15, 30 and 60 min of gravistimulation andexposure to CMF. The samples were post-fixed with 1%(w/v) OsO4 at ambient temperature. Samples were dehy-drated in ethanol and acetone series and embedded inepon-araldite. Thin (1.7–1.9 lm) sections were obtainedwith an ultramicrotome MO-XL (RMC) and subjected toPeriodic Acid Schiff reaction and stained with toluidineblue and examined in an AXIOSCOPE (Zeiss) with imagePhoto software.

2.3. Calcium ion distribution

Seedlings were incubated in the calcium-specific fluores-cent dye fluo-4 (Molecular Probes, USA) for 25 min in

darkness prior to treatments in the CMF. The stock solu-tion of indicator was prepared in the concentration of1 mM in anhydrous dimethylsulphoxide (DMSO) asdescribed (Haugland, 2002). The final concentration ofindicator loading solution was 8 lM with a final volumeof 1 ml.

Observations were carried out with a confocal laserscanning microscope LSM 5 Pascal (Zeiss) at the excitationwavelength 494 nm and emission wavelength 516 nm. Thedistal elongation zone of a length �400 lm (at �800 lmfrom the root apex) was scanned across a root in the direc-tion from the upper side to the lower side of a gravistimu-lated root. Calcium ions distribution and quantification inthe cells were obtained using software ‘‘Pascal’’, whichallows for color-encoding of the fluorescence intensity ofcalcium ions after treatment with the indicator. The inten-sity of the fluorescence correlates directly with the concen-tration of cytosolic calcium at the corresponding emissionwavelength (McAinsh et al., 1992, 1995; Franklin-Tonget al., 1997; Ng and McAinsh, 2003).

3. Results

3.1. Microscopic analysis

Results from light-microscopy images of the columellacells from roots with either a positive or a negative gravi-tropic reaction (Fig. 1) supported data obtained earlier(Kordyum et al., 2005) showing that after 60 min of grav-istimulation, all amyloplasts sedimented in the distal partof control columella cells in controls whereas for plantstreated with the CMF, they were localized close to one ofthe longitudinal walls. After 15 min of gravistimulation,amyloplasts displaced to the physically lower side of a rootin the control columella, but in the CMF treated plants,they did not sediment to the direction of a gravitationalvector and were distributed over all the cell volume. After30 min of gravistimulation in the CMF, amyloplasts tendedto aggregate towards the center of the statocytes, oftenscattered along the long axis of cell (the position of amy-loplasts was similar to that found in microgravity) (Kord-yum et al., 2005).

Under gravistimulation in the CMF, the nucleus in thestatocytes stayed within the proximal region similar tothe control. The distribution of actin filaments aroundnucleus and amyloplasts was similar in statocytes of bothcontrol and experimental columella cells (Kordyum et al.,2005).

3.2. Lateral distribution of calcium ions in the root distal

elongation zone

A growth region in the elongation zone, where cellselongate slowly, located between the apical meristem andthe distal portion of the region of rapid cell elongation isnow defined as the transition zone. Cells of this zone arein a developmental transition from cytoplasmically driven

Table 1Fluorescence intensity of calcium ions bound with specific fluorescentcalcium indicator fluo-4 in the DEZ after 10 min of gravistimulation, rel.units

Conditions ofgravistimulation

DEZ Seedlings grown in

Water Water withCaCl2

SMF Firsthalf

Upper 115.91 ± 3.31 149.75 ± 2.43Lower 184.43 ± 5.51 230.59 ± 3.47

Secondhalf

Upper 130.59 ± 2.26 72.37 ± 1.13Lower 194.59 ± 3.08 221.05 ± 2.86

CMF Firsthalf

Upper 155.07 ± 4.07*** 232.83 ± 3.45***

Lower 116.24 ± 2.36*** 192.26 ± 2.98***

Secondhalf

Upper 159.32 ± 3.53*** 193.77 ± 2.67***

Lower 100.84 ± 2.41*** 128.39 ± 2.23***

Mean ± SE; ***p < 0.001 for all values.‘‘Upper’’ and ‘‘lower’’ mean upper and lower sides of the gravistimulated

root.

Fig. 1. Cress seedlings after 30 min (a and c) and 1 h (b and d) of gravistimulation in SMF (a and b) and in CMF (c and d).

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expansion to vacuome driven elongation. Cells traversingthe transition zone use cytoskeletal elements to regulateboth growth polarity and the maintenance of cellulargrowth (Baluska et al., 1990). Cortex and epidermis cellsshow a prominent transition zone composed of around25 cells in each longitudinal cell file – ‘‘postmitotic isodia-metric growth zone’’ (Baluska et al., 1990). We use the termdescribed by Ishikawa and Evans (1995) for this regioncalled ‘‘distal elongation zone’’ (DEZ). This terminologyindicates that these cells are near the distal end of the elon-gation zone but does not categorize them in terms of mito-tic activity, shape, or allometric coefficient of expansion.The main distinguishing feature of these cells is their specialphysiological properties. The cells in DEZ differ from cellsof the main or central elongation zone (CEZ) with respectto auxin sensitivity and DEZ cells play an important role ingravitropism. DEZ cells are also sensitive to other diverseendogenous clues and exogenous factors, such as ethylene,extracellular calcium, mechanical pressure, water and saltstress, gravity, aluminium, and microorganisms (Ishikawaand Evans, 1993, 1995; Baluska et al., 1994, 2001; Fasanoet al., 2001).

Calcium ions in the presence of specific fluorescent indi-cators are brightly fluorescent in green color in the root epi-dermis and cortex layers. We present the averagefluorescent intensity in the first half (200 lm) of DEZ,beginning from the meristem, and the second half(200 lm) of DEZ (Tables 1–3). Each half of DEZ wasscanned twice over the distance of 100 lm. After 10 minof gravistimulation, in the first half of DEZ, the ratiobetween fluorescence intensity on the lower to upper sidesof the gravistimulated root was 1.6 in the control, staticmagnetic field (SMF; Table 1, Fig. 2a and g) whereas the

ratio between fluorescence intensity on the lower to upperof the gravistimulated root in CMF was 0.8 (Table 1,Fig. 2d and j). When seedlings were grown in water withCaCl2, these ratios for the first half of the DEZ for SMFand CMF were 1.5 and 0.8, respectively (Table 1). In thecontrol, in the second half of DEZ, the ratio betweenfluorescence intensity on the lower to upper sides of thegravistimulated root for water was 1.5 (Table 1). For thesecond half of the DEZ for roots from the CMF, the ratiobetween fluorescence intensity on the lower to upper sidesof the gravistimulated root was 0.6 (Table 1). For thesecond half the DEZ for plants treated with water andexternal calcium the ratios of lower to upper side intensitywere 3.1 and 0.7 for the control and CMF treated plants,respectively (Table 1).

Table 3Fluorescence intensity of calcium ions bound with specific fluorescentcalcium indicator fluo-4 in the DEZ after 30 min of gravistimulation, rel.units

Conditions ofgravistimulation

DEZ Seedlings grown in

Water Water withcalcium

SMF Firsthalf

Upper 119.48 ± 2.39 160.58 ± 2.54Lower 145.82 ± 3.75 215.31 ± 3.02

Secondhalf

Upper 92.96 ± 2.16 119.07 ± 1.54Lower 138.92 ± 2.87 214.19 ± 2.74

CMF Firsthalf

Upper 186.63 ± 3.33*** 227.90 ± 3.13***

Lower 112.94 ± 2.25*** 170.33 ± 2.44***

Secondhalf

Upper 149.95 ± 3.53*** 202.19 ± 2.83***

Lower 79.23 ± 2.01*** 127.49 ± 1.69***

Mean ± SE; ***p < 0.001 for all values.‘‘Upper’’ and ‘‘lower’’ mean upper and lower sides of the gravistimulated

root.

Table 2Fluorescence intensity of calcium ions bound with specific fluorescentcalcium indicator fluo-4 in the DEZ after 20 min of gravistimulation, rel.units

Conditions ofgravistimulation

DEZ Seedlings grown in

Water Water withcalcium

SMF Firsthalf

Upper 99.04 ± 3.00 102.41 ± 1.96Lower 169.10 ± 4.09 174.85 ± 2.72

Secondhalf

Upper 74.48 ± 2.28 102.30 ± 1.31Lower 164.64 ± 3.04 202.43 ± 2.52

CMF Firsthalf

Upper 181.69 ± 3.40*** 220.29 ± 3.12***

Lower 107.80 ± 1.69*** 153.35 ± 2.18***

Secondhalf

Upper 126.78 ± 3.53*** 195.44 ± 2.71***

Lower 66.57 ± 1.36*** 119.12 ± 1.97***

Mean ± SE; ***p < 0.001 for all values.‘‘Upper’’ and ‘‘lower’’ mean upper and lower sides of the gravistimulated

root.

E. Kordyum et al. / Advances in Space Research 39 (2007) 1210–1217 1213

After 20 min of gravistimulation, in the first half ofDEZ, the ratio of intensity from lower to upper for SMFand CMF for water treated samples was 1.7 and 0.6,respectively (Table 2, Fig. 2b, h, e and k). In the presenceof external Ca2+, the ratios of lower to upper intensity were1.7 and 0.7 for the SMF control and CMF roots, respec-tively. In the second half of DEZ, this ratio increased to2.2 for the SMF roots and reduced to 0.5 for the CMF trea-ted roots. With the external calcium, the ratios for the SMFand CMF of the second half of the DEZ roots were 2.0 and0.6, respectively (Table 2).

After 30 min of gravistimulation, in the first half ofDEZ, the ratio between fluorescence intensity on the lowerto upper sides of the gravistimulated root reduced to 1.2when compared to both previous time intervals for theSMF treated roots (Table 3, Fig. 2c and i). The ratiobetween fluorescence intensity on the lower to upper sidesof the gravistimulated root in CMF at 30 min (0.5) wassimilar to that for 20 min interval (0.5) but was reduced

compared to that after 10 min of gravistimulation (0.6)(Table 3, Fig. 2f and l). When external calcium was addedin the water, the ratio (lower/upper) of the first half of theDEZ in regard to the conditions of SMF decreased to 1.3in comparison with previous time intervals but for CMFtreated roots this ratio fluctuated over the course of thetime treatments (Tables 1–3). For controls of the secondhalf of DEZ with water, the situation was similar to thatin the first half of DEZ, the ratio diminished to 1.5 inSMF (Table 3) This ratio was 0.5 for the CMF treatedplants (Table 3). After 30 min, for the second half of theDEZ, roots treated with external calcium had ratios oflower to upper intensity of 1.8 and 0.6 for SMF andCMF treated plants, respectively (Table 3).

4. Discussion

4.1. The CMF-induced Ca2+ resonance effects ingraviresponse of the roots

Our report (Kordyum et al., 2005) was the first to dem-onstrate that CMF with the cyclotron resonance frequencyof calcium ions affects root gravitropism. It was shown thatgravistimulation of cress roots in this field changes the nor-mal positive gravitropic response to a negative one.

A number of biological resonance frequency-dependenteffects of combined magnetic field have been demonstrated(Liboff, 1985; Liboff et al., 1987; Blanchard and Blackman,1994). CMF is the sum of parallel BDC and alternating BAC

magnetic fields:

B ¼ BDC þ BAC cosð2pftÞ; ð1Þ

where f is the frequency of alternating field. Usually theamplitudes BDC and BAC were of an order of geomagneticfield of �50 lT. It was found that the frequencies of theseresonance effects can be calculated by the following:

fc ¼ qB=2pm; ð2Þ

where q is the ion’s charge (q = 2e for Ca2+ ion), e is elec-tron’s charge, m is ion mass, B is magnetic induction. Res-onance effects were also observed at some higher oddharmonics of this frequency (McLeod et al., 1987a,b; Smithet al., 1987). Since formally the Eq. (2) corresponds to socalled ‘‘cyclotron frequency’’ of ions, the changes in func-tional properties of biosystem at the frequency fc have beencalled ‘‘ion cyclotron resonance in biosystems’’ (Liboff,1985).

If we add an oscillating magnetic field collinear to a per-manent one with the frequency that coincides with fc, theenergy will be transferred from alternating field to ions ina resonance manner. This will cause the ions to move morerapidly. Cyclotron resonance may be produced any timethere is a steady magnetic field combined with an oscillat-ing electric or magnetic field acting on a charged particle.The (Eq. (2)) shows that for an average strength of theEarth’s magnetic field the frequencies for the oscillatingfields that are needed to produce resonance with the biolog-

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ically important ions is approximately 0–100 Hz and this isthe most significant part of our electromagneticenvironment.

Although the model of ‘‘ion cyclotron resonance in bio-systems’’ (Liboff, 1985) seemed to explain the resonanceCMF effects, it did suggest that this model has some prob-lems. These include: (1) problems with viscosity of a liquidmedium, the real cyclotron motion of ions in liquids cannottake place even in rather high magnetic fields because of

Fig. 2. Fluorescent intensity of calcium ions in the first half of a root distal10 min of gravistimulation (a,g: SMF; d,j: CMF), 20 min of gravistimulation (b

ion hydrate shell; (2) the huge magnitude (about a meter)of the radius of free Ca ion rotation at room temperature.These difficulties were avoided by the suggestion that cyclo-tron resonance is developing inside the vacuum within ionchannels. In such a case there will be an extremely smallnumber of ions acting at the very low-energetic part ofMaxwellian distribution tail (Engstrom, 1996).

Besides ion cyclotron resonance model (Liboff, 1985)there have been models of ion parametric resonance

elongation zone visualized with confocal laser scanning microscopy after,h: SMF; e,k: CMF), and 30 min of gravistimulation (c,i: SMF; f,l: CMF).

Fig. 2 (continued)

E. Kordyum et al. / Advances in Space Research 39 (2007) 1210–1217 1215

(Lednev, 1991, 1996), and its variation (Blanchard andBlackman, 1994). The last model is identical to Lednev’smodel except for an assumption that the forces, whichthe AC and DC magnetic fields exert on the moving ion,obey different physical laws. The detailed analysis of basicassumptions of Lednev’s model was carried out by Eng-strom (1996). He took into account the role of the noiseand showed that despite the difficulties the main sugges-tions of Lednev’s model are not contradictory and themodel gives clear predictions to be tested experimentally.According to Lednev (1991) a combination of DC andAC applied magnetic fields changes the transition probabil-ity between different vibrational energy levels of ion–pro-tein complex. If an ion–protein complex can be indifferent energy states which have different biochemicalactivities that effect developmental and growth processesthen the CMF may alter biological responses by changingthe ion–protein energy state. The theory proposed by Led-nev (1991, 1996) shows that magnetic fields of the order oftenths of a lT (i.e. much lower than the energy of thermalmotion) are sufficient to produce biological effects. Thismodel has been successfully tested for Ca2+ and K+ ionsby Prato et al. (2000), Yost and Liburdy (1992) and others.

The gravistimulation of cress roots in the CMF with dif-ferent frequencies showed that (1) the direction of a gravi-

tropic reaction depends on the relationship between theparameters of the CMF such as magnitudes BAC and BDC,their ratio BAC/BDC and the frequency f of alternating mag-netic field; (2) under some sets of these parameters, there isthe resonance response of the roots bending to the character-istics of the CMF; (3) the dependence of root graviresponseon the frequency of alternating magnetic field has a reso-nance peak at f = fc that is equal to the cyclotron frequencyof Ca2+ ions for the magnetic field BDC.

4.2. A root gravitropic reaction in the CMF

Based on the analysis of the data we propose that rootgravitropic responses in the CMF occurs by the normalphysiological process resulting in root bending, althoughin an opposite direction (Table 4).

There are changes in amyloplast behavior and Ca2+ lat-eral redistribution in a gravistimulated root in the CMF.Data from recent experiments support the function of amy-loplasts as statoliths and also consider plastid grouping as amechanism that affects gravisensitivity (Smith et al., 1997).Amyloplasts in columella cells in the CMF were alsoenmeshed within actin microfilament network and we didnot find obvious differences between control and experi-ments in this respect. Behavior of amyloplast in the CMF

Table 4A proposed model for root gravitropism under normal conditions (positive gravitropism) and in CMF treated roots (negative gravitropism)

Response Root positive gravitropism Root negative gravitropism (CMF)

Displacement of amyloplast

15 min after gravistimulation Movement of statocytes Movement of statocytes30 min after gravitstimulation Sediment on distal side of statocyte Aggregate towards center of statocyte60 min after gravistimulation Sediment on distal side of statocyte Localize to one of the longitudinal walls of the statocyte

Statoliths enmeshed in microfilament network Yes YesCa2+ redistribution Lower side of gravistimulated root Upper side of gravistimulated rootPolar auxin transport Yes Unknown

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has been discussed more detail in the previous paper(Kordyum et al., 2005).

More calcium accumulation was documented in the epi-dermis and cortex cells of the concave side of a gravistimu-lated root in the control and in the CMF whichcorresponded to hypothesis that high concentrations ofextra cellular Ca2+ in growing tissue can result in substan-tial inhibition of growth (Hasenstein and Evans, 1986). Inaddition, there is evidence that supports some involvementof Ca2+ ions in gravitropism. It was shown that treatmentof plant tissues with calcium ionophore A23187 which arti-ficially elevates ½Ca2þ�i resulted in the disruption of boththe polarity of statocytes and subsequent gravisensing(Wendt and Sievers, 1989). However, there is also evidencethat Ca2+ may not be directly related to gravitropism(Hasenstein and Evans, 1988; Legue et al., 1997).

Gravistimulation has been shown to initiate rapid Ca2+

movement to the lower part of a root or to the upper partof a shoot, so the concentration of wall Ca2+ is higher inthe region of lower growth and the opposite is true forregions of accelerated growth ( Lee et al., 1983; Moore,1985). In the CMF, the Ca2+ concentration is higher inthe upper side of a gravistimulated root, and a gravitropicreaction is negative. In addition, a ratio between fluores-cent intensity on the lower to upper sides of a gravistimu-lated root in the CMF was lower than this ratio in thecontrols, and it also increased when seedlings grown inwater with CaCl2. Calcium added in the external mediumsupports more striking response to gravistimulation inboth SMF and CMF with more pronounced Ca2+

redistribution.It should be noted that in the second half of DEZ, the

lateral redistribution of calcium ions was more strikingthan in the first half of DEZ regardless of the conditionsof gravistimulation as well as the conditions of seedlingsgrowth. In both halves of DEZ, the most prominentCa2+ redistribution was revealed after 10 and 20 min ofgravistimulation in the control (SMF) independently ofthe conditions of growth. In CMF, the most remarkableCa2+ lateral redistribution was observed after 30 min ofgravistimulation.

CMF conditions increase the Ca2+ lateral redistributionto the upper sides of a gravistimulated root, so we canhypothesize that in the CMF with the alternating compo-nent adjusted to the cyclotron frequency of Ca2+ (Liboff,

1985), the energy is transferred from alternating compo-nent to ions in resonance manner. This resonance causesthem to change the parameters of their movement, in par-ticular from a root cap columella where the high levels ofCa2+ around amyloplasts have been demonstrated (Buschet al., 1993). It is also possible that there are alterationsin ion ‘‘sensitivity’’ to the direction of a gravitational vectorunder gravistimulation in the CMF. Bioelectrical gradients,which are suspected to be induced by gravistimulation ofaxial organs and result from transport of Ca2+ and H+ ionsacross membranes (Slocum and Roux, 1983), may alsochange in CMF.

Thus, the results presented here demonstrate that bothamyloplasts and Ca2+ ions are involved in plant gravitro-pism. However, further studies are warranted to answersome important questions about gravitropism. (1) Are thedifference in wall Ca2+ is sufficient to alter growth asymmet-rically? The observed differences may be sufficiently large tocause unequal auxin transport and growth and thus initiatebending (Sinclair and Trewavas, 1997). The presence of twomotors driving root gravitropism is assumed (Wolvertonet al., 2002; Perrin et al., 2005), one of which appears notto be auxin regulated. (2) What are possible mechanismsof Ca2+ movement in response to gravistimulation, espe-cially with respect to the formation of a lateral asymmetryin apoplastic distribution of these ions (Slocum and Roux,1983)? (3) What force or forces cause the amyloplast unu-sual displacement under gravistimulation in CMF? (4)What is a role of Ca2+ ions in a sign change of root gravit-ropism – primary or secondary (inhibition of cell expansionin the distal elongation zone)? (5) What is Ca2+ function inthe statocytes in CMF?

Using the model of a root negative gravitropic reactionin CMF with the frequency resonance to the cyclotron fre-quency of Ca2+ ions will be effective for future research toidentify the mechanism of plant gravitropism, including arole of Ca2+ in other plant growth responses.

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