J. Biol. Macromol., 5(2), 31-38 (2005)

Effect of functional water treated with materials irradiating far-infrared light on theorientation of cortical microtubules in Spirogyra cells
Kazuyoshi Iwata and Yoh Sano

Faculty of Pharmaceutical Sciences, Setsunan University(Nagaotoge-cho, Hirakata, Osaka, 573-01, Japan)
Received March 14, 2005, accepted May 11, 2005.
Effect of functional water on living organisms was investigated by using an alga,

Spirogyra. The orientation of cortical microtubules (MTs) in Spirogyra cells
repolymerized in functional water I containing 50mM NaCl treated by HIET plate,which irradiates

far-infrared light [Nihon Reitou Yusou], after depolymerization withAmiprophos-methyl (APM), was different from that of cortical MTs repolymerized in 50
mM NaCl. Functional water I affected the orientation of cortical MTs in the same
direction as the decrease of NaCl concentration. Functional water II in the presence
50mM NaCl

prepared by diluting 1M NaCl with the DW treated by HIET plate alsosimilarly affected the orientation of cortical microtubules (MTs) to functional water I.
These results suggest that Spirogyra cells could be useful for investigating the effect offunctional water on living organisms.
Key words: Spirogyra, cortical microtubules, functional water, far-infrared light, salt
damageRecently,

somewatergivensomeenough evidence that insures utility offunction by treating with a weak energy,functional water.

so-called functional water, is used in wideof estimating effects of functional water

fields (1). Especially, in agriculture oron living organisms in a short time islivestock industry, functional water isnecessary.

Experimental systemused for increase of yield, improvement ofIn plant cells, cortical microtubules

quality and so on. However, there is not(MTs) exist just beneath the plasma
membrane. The orientation of corticalThe

abbreviations

APM,

MTs was found to be parallel to theArtificial

orientation of cellulose microfibrils in

pond water; MT, microtubule; ORP

some cases. Therefore, it is believed thatoxidation-reduction potential.

cortical MTs play important role in cell

Amiprophos-methyl;

used

are:

APW,

32

Effect of functional water on cortical microtubules

Spirogyra,aSpirogyra fluviatilis Hilse (Fig. 1a) wasspecies of algae, the orientation of corticalisolated from a pond in Joyo, Kyoto. It

MTs is affected by constitution of ions

was cultured in Ichimura medium (10)(3,4), change of turgor pressure (5) and sounder the light conditions of 16-h lighton (4). In addition, detectable change ofand 8-h darkness at 250C. Cells were

the orientation of cortical MTs appears

used for the experiments within 2 to 7

within 90 min. These facts predict thatdays (growing stage) after transferring

subtlethem from the stock culture to the freshmorphogenesis(2).

changesInneartheplasma

membranes induced by water treatment

Ichimura medium.

may be detected in a short time. In theArtificial pond water (APW) (0.1mM

present study, the effect of functional

each of KCl, NaCl, CaCl2 and 5mM

water on the cortical MTs in Spirogyra

Hepes-Tris [pH7.5]) was used as thecells is studied and then the possibility

experimental medium when cortical MTs

that Spirogyra cells could be used forin Spirogyra cells were depolymerized.

evaluating functional water is examined.

Amiprophos-methyl (APM) was dissolved

The orientation of cortical MTs in

in DMSO to a concentration of 5mg/ml.

Spirogyra cells is affected by Na+ (3, 4).

Cells were treated with 3μg/ml APM in

High Na+ concentration can be harmful toorder to depolymerize the cortical MTs forsome of fresh water algae and land plants

24 h. Then the APM was removed byandphysiologicalwashing the cells quickly three times

processes (6,7,8,). Accumulation of salt inwith distilled water. Cortical MTs weresoil is one of the most serious problemreorganized in various concentrations of(salt damage) inhibiting agriculture inNaCl or functional water for 1.5 h. Whenthesalteffects of control water and functional

composition of saline soils is NaCl (9). If

water were compared, the concentration

functional water regulating the effect ofof NaCl was adjusted to 50mM because

Na+ exist, it may be useful for lessening

MT orientation changes sensitively tosalt

weNaCl concentration around 50mM (seefunctionalwaterFig. 2). 50mM NaCl was prepared byNa+on theinterferesworld.

Thedamage.

investigatedwithmostIn
howthisinfluences the effect ofcommonstudy,diluting 1M NaCl with DW.

orientation of cortical MTs.

Preparation of functional waterMATERIALSANDMETHODS

Functional water I was prepared byplacing 50mM NaCl solution on HIET

Plant materials and treatments withplate (Nihon Reitou Yusou) irradiating

solutionsfar-infrared light (4 to over 20 μK. Iwata and Y. Sano33m[personal communication]) for 10min.

p-phenylenediamine

Functional water II was prepared by

The orientation of MTs was determinedplacing DW on HIET plate for 10 min andusingthen diluting 1M NaCl with the DW toOrientation of MTs was classified into

50mM NaCl.

three categories based on MT angles toandihydrochloride).

epifluorescencemicroscope.

the cell axis, longitudinal (L) for 00-100,

Immunostaining

oblique (O) for 100-800, transverse (T) for

MTs were observed using the method of800-900.

Oblique(O)wasfurther

Hogetsu (11). Cells were fixed in 3.7%classified into S-helix and Z-helix, which

formaldehyde

are indicated as SO and ZO, respectively

phosphatein50buffermM(pH7.0)

sodiumatroom(see Fig.3).

temperature for 30 min. They were thenResponse of cells is slightly different in

cut in the same fixative with a razor

each experiment. MT orientation of cells

blade to allow entry of antibodies. Cells

treated with control water and functional

were washed three times with 50mM

water was observed at the same time insodium phosphate buffer and treatedeach experiment.

with a detergent solution (1% Triton
X-100 and 0.4M mannitol in 50mM
sodium phosphate buffer [pH7.0]) for 30

Statistical Analysis
Statistical analysis was performed with
Wilcoxon two-way analysis of variance

min at room temperature.
After removal of the detergent, they

followed by two-sample Wilcoxon test.

were incubated with mouse monoclonal

Z0>1.96 was considered as statisticallyantibody against chick brain α-tubulin

significant.

(Sigma,T9026)

diluted1:250

inphosphate-buffered saline (PBS, pH7.3)

Measurement of pH, conductivity andat room temperature, overnight. They

oxidation-reduction potential (ORP)

were washed three times with 50mM

pH of solutions was measured bysodium phosphate buffer and stained atpH/IONroomwith

Conductivity and ORP of solutions wereFITC-conjugated antibodies raised inmeasured by pH/Cond METER D-24

sheep against mouse IgG (Sigma, F2883)(Horiba, Ltd.).

temperaturefor3hmeterF-23(Horiba,Ltd.).

diluted 1:200 in PBS.
Cells were washed three times withRESULTS

ANDDISCUSSION

50mM sodium phosphate buffer andmounted with a solution (50% glycerol inPBS

containing

0.1%

Cortical MTs in Spirogyra cells are34

Effect of functional water on cortical microtubules

abcdFig. 1 Photographs of Spirogyra cells and immunofluorescence images of cortical MTs in

Spirogyra cells. a, Spirogyra cells. b, Transverse (100-800 to the cell axis) MTs. c, oblique(100-800 to the cell axis) MTs. d, longitudinal (00-100 to the cell axis) MTs. Bars=30μm.

100Frequency (%)

80
TSO
ZO
L

60
40
20
00

25
50
75
100
Concentration of NaCl (mM)

Fig. 2 The orientation of cortical MTs repolymerized in various concentrations of NaCl.
T, SO, ZO and L indicate the cells having transverse, S-helix, Z-helix and longitudinalMTs, respectively. In one experiment, about 20 cells were examined and 5 experiments
were repeated.
transverse in normal condition (Fig. 1b).
However,theof

Functional water prepared by placing

after

50mM NaCl solution on HIET plate fortreatment of anti-MT agent, APM, for 24

10min (functional water I) or control

h, changes from oblique (S-helix), obliquewater (50 mM NaCl in DW) were used as(Z-helix)

repolymerized(Fig.

longitudinalorientation(4).

MTsinNaCl1c)andthentosolutions(Fig.

1d)

asthe

depolymerized MTs. In one experiment,

concentration of NaCl increases (Fig.2, 3)

forreorganization

20 cells were examined and theoftheK. Iwata and Y. Sano

35

increase of NaCl （Na＋） concentration
Fig. 3 Relationship between NaCl
concentration and the orientation ofcortical MTs

S-helix

Z-helix

longitudinal

Frequency(%)

60

40

TSO
ZO
Lothers

2020x20050mM NaCl

Functional Water I

Fig. 4 The orientation of cortical MTs repolymerized in 50mM NaCl and functional
water I. T, SO, ZO and L indicate the cells having transverse, S-helix, Z-helix andlongitudinal MTs, respectively. In one experiment, 20 cells were examined and 20experiments were repeated.
orientation of cortical MTs in 400 cells in

direction shown by arrows in Fig. 3 weretotal was decided in 20 experiments. In

notcells treated with functional water I, thefunctional water. Conductivity of solution

ratio of cells having longitudinal MTs

did not change by HIET treatment (Table

decreased and that of cells having oblique1). These results imply that functional(S-helix) MTs increased, compared withwater I could lessen the effect of Na+. pH

cells treated in control water (Fig. 4).

and ORP did not change, either (Table 1).

Statistic analysis showed appreciable

Na+ is known as an element inhibiting

differences.

experiment,growth of some of fresh algae and landsignificant differences were observed inplants (salt damage) (6,7,8). Salt damage

10 experiments but not in the other 10

is one of the most serious problem

experiments. However, the changes of thelimitingorientation

products. This species of Spirogyra is also

Inof

each

corticalMTsintheobserved

theafteramounttreatmentofwithagricultural

36

Effect of functional water on cortical microtubules

Table 1 pH, Conductivity and ORP of each solution. They were measured three times.

ｐH50mM NaCl
Functional Water I
Functional Water II

conductivity (mS/cm) ORP(mV)

5.48+0.14
5.47+0.12
5.43+0.07

5.68+0.07
5.65+0.08
5.71+0.11

348+3347+3348+3Na+-sensitive. Many cells died after

experiment, 20 cells were examined andincubation for 24 h in 100mM NaClthe orientation of cortical MTs in 200solution (4). The present results maycells

indicate a possibility that HIET-treatedexperiments. In the case of cells treatedwater may lessen salt damage without

in functional water II, the ratio of cellschange of ionic strength.

having longitudinal MTs decreased andintotal

wasdecided

in10

Next, functional water II, which wasthat of cells having oblique (S-helix) MTs

prepared by placing DW on HIET plateincreased, compared to the case of cells

for 10 min and diluting 1M NaCl bytreated in control water (Fig. 5). Statistic

HIET-treated DW to 50mM, was also

analysisexamined.

differences existed. In each experiment,

DepolymerizedMTs

wereshowedthatappreciable

reorganized in functional water II or

significantcontrol water (50mM NaCl in DW). In one

experiments but not in the other 6differences

existedin4Frequency(%)

60

40TSOZO
Lothers

2020x10050mM NaCl

Functional Water II
Fig. 5 The orientation of cortical MTs repolymerized in 50mM NaCl and functional
water II. T, SO, ZO and L indicate the cells having transverse, S-helix, Z-helix andlongitudinal MTs, respectively. In one experiment, 20 cells were examined and 10experiments were repeated.

K. Iwata and Y. Sano37experiments. However, there was noaffecting the orientation of corticalexperimental result that the orientation

microtubules in selected plant cells.

of cortical MTs after treatment withDoctoral thesis for Kyoto University

functional water II change significantly

5. Iwata, K., Tazawa, M., and Itoh, T.

in the direction shown by arrows in Fig. 3,(2001) Turgor pressure regulation andwhich

thewassimilartothecaseoforientationofcortical

functional water I. This result may imply

microtubules in Spirogyra cells. Plant

that HIET-treated DW also keeps theCell Physiol. 42:594-598.

similar function to HIET-treated 50mM

6. Katuhara, M. and Tazawa, M. (1986)

NaCl. As mentioned in materials andSalt tolerance in Nitellopsis obtuse.

methods, response of cells is slightly

Protoplasma 135: 155-161.

different in each experiment. Frequency

7. Sakamoto, A., Murata, A. and Murata,of cells in 50mM NaCl in Fig. 5 was

N. (1998) Metabolic engineering of rice

different from that in Fig. 4.

leadingThe

present

studysuggests

thatandwhether functional water differs from1011-1019.

functional water on living organisms.

biosynthesis

ofglycinebetaine and tolerance to salt

Spirogyra cells are useful for judging
control water and inferring effects oftocold.

PlantMol.

Biol.

38:

8. Mitsuya, S., Takeoka, Y. and Miyake, H.
(2000) Effects of sodium chloride on
foliar ultrastructure of sweet potato(Ipomoea

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Communicated by Yoshihiko Amano.