DEVELOPMENTAL & REPRODUCTIVE BIOLOGY ISSN1004-6453/CN11-3569/Q

Vol. 9 No. 1 pp: 65-72 Jun. 2000 2000 Chin. Dev. & Reprod. Biol. Socs.
Vol. 9 No. 1 (2000) 65
Electrical Characters on Surface of Apoptotic
Cell in Maize Roots Induced by Cytotoxins
1

NING Shunbin, SONG Yunchun
2
and WANG Ling
Center for Developmental Biology, Wuhan University, Wuhan 430072, China

ABSTRACT The difference of electrophoretic mobility (EPM) value between normal
and apoptotic cells induced by cytotoxins cycloheximide, actinomycin D and colchicine
was firstly identified using the method of cell electrophoresis. Combined with the
protoplast preparation, the change of electric charge on protoplast membrane in
apoptotic cells was quantified directly in an in vivo system of the root tip of maize.
Experimental results indicated that the absolute value of Zeta potential on the
membrane of apoptotic plant cells induced by cytotoxic drugs was much higher than
that in normal cells. The presented results prompted that the method of cell
electrophoresis could be thought as a creative approach to accomplish quantitative
analysis of surface electric charge of cell membrane of apoptotic cells.
Keywords: cell electrophoresis, plant apoptosis, cytotoxic drugs, chromosome
preparation
1. INTRODUCTION
In animal, the induction of apoptosis by cytotoxins RNA-synthesis inhibitor actinomycin D
(ActD), protein-synthesis inhibitor cycloheximide (CHX) and mitosis inhibitor colchicine
(COL) was reported earlier and all of them resulted in DNA ladder, which was showed by
gel electrophoresis. Although the apoptosis processes induced by these cytotoxins showed

1
The authors want to thank Dr. Maki Katsuhara (Okayama University, Japan), Dr. Blazena
Koukalova (Academy of Sciences of the Czech Republic, CZ), Dr. Iona Weir (Horticulture and Food
Research Institute of New Zealand, new Zealand), Dr. Hong Wang (University of California, USA)
and Dr. Patrick Gallois (University of Perpignan, France) for their kind sending data concerned and
valuable suggestions.
This project is granted by State natural Science Foundation (Grant No.39870423) and State
Commission of Education, Doctorate Spot Fund.
2
Author for correspondence. Tel: (027)87684505. E-mail: ycsong@whu.edu.cn.
NING Shun-bin et al.
66 Developmental & Reproductive Biology
different initiating mechanisms but they involved a common regulation mechanism
[1-5]
.
There is increasing evidence that as well as in animals, a large number of physical or
chemical factors can induce apoptosis-like cell death in plants besides the convincing
examples which have been well described using in vitro cultured tissues or
suspension-cultured cells induced by UV-B irradiation
[6]
, cold stress
[7]
, KCN
[8]
, and heat
shock
[9]
, and using root meristematic cells induced by salt stress
[10]
and by mannose
[11]
.
There are also some instances of identification of apoptotic characteristics in plants
responded to cycloheximide (CHX)
[12]
and other toxins
[8,13,14,15]
. Recently, using the root
tip of maize as experimental material, a research regarding the induction of apoptosis in cells
was accomplished and found that those cytotoxic drugs could also induce typical apoptotic
characteristics, as chromatin condensation, nucleus degradation as well as occurrence of
DNA ladder in the cells of root tip of maize
[16]
.
The event of specific changes in cell surface is one of the characters of apoptosis in animal
cells
[17]
. It was found that the entire course of apoptosis is accompanied by physical and
chemical changes of cell membrane. During the early events of the signal transduction,
some biochemical changes of plasma membrane such as the increased exposure of acidic
phospholipid phosphatidylserine (PS) have been detected. Even there could be observed
the loss of microvilli through electron microscopy. Interestingly, there also happen the
corresponding membrane events in apoptosis in plant cells. For instance, the occurrence of
ion leakage which resulted from increase of membrane permeability in hypersensitive
response (HR) in pathogen-infected plants provides a basis for counting the number of
apoptotic cells
[18]
. Dead cells will finally lost their function of plasma membrane, which can
be used to monitor the cell death
[19]
. Recently, apoptosis in plants induced by some drugs
showed an enrichment of PS on the outer surface of the plasma membrane as detected by
fluorescein-conjugated annexin-V binding.
[15,20,21]
. Plasma membrane alteration is an early
indicator of cells undergoing apoptosis since it occurs prior to the detection of DNA strand
breaks by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling
reaction (TUNEL)
[6]
. So it is well worthy of investigation.
We infer that these changes of cell surface during apoptosis process probably cause variation
in Zeta potential on cell surface. Since the cell electrophoresis technique applied in cells is
referred to one of the important approaches for researching the state of electric charge on cell
surface, it is applied in apoptosis in of maize root tips induced by these cytotoxins mentioned
above.
Electrical Characters on Surface of Apoptotic Cell
Vol. 9 No. 1 (2000) 67
2. MATERIALS AND METHODS
2.1. EXPERIMENTAL MATERIALS AND CHEMICALS
The tested seeds of maize, derived from an inbred line Huang Zao 4, were denoted by Prof.
Song Jiancheng, Shandong University of Agriculture, China. The seeds were soaked in
double-distilled water for 1 d, and, after germinating, were transplanted in a chamber with
culture medium (consisting of 4 mmol/l KNO
3
, 1 mmol/l NaH
2
PO
4
, 1 mmol/l MgSO
4
, 1
mmol/l CaCl
2
, 1 mmol/l Fe-Citrate, pH 5.5 adjusted with NaOH) for successive culture at 28
in the dark. When the root tips were around 1.5 cm in length, the medium was adjusted
to a final concentration of 0.1 mg/l with above chemical mixture, and the culture lasted for
an appropriate period of time under dark condition
[10]
.
The cytotoxins, actinomycin D (ActD), cycloheximide (CHX) and colchicine (COL), were
purchased from SERVA Co.
2.2. PREPARATION OF PROTOPLAST SUSPENSION
Root tips after treatment and control of around 0.5 mm were cut from the germinating seeds
at different time points and fixed immediately with 4% paraformaldehyde at 4 overnight.
After that, the root tips were digested with DNase-free enzyme mixture (2% cellulase, 1%
pectinase, SERVA) in 6.8 mmol/l CaCl
2
, 11 mmol/L KH
2
PO
4
, 0.6 mol/l mannitol, 4g/l
polyvinylpyrrolidone (PVP) (pH5.8) at 28 for around 3 h. After enzymolysis, puffed the
softened tips slightly with a sucking tube to let a large amount of protoplast be dissociated.
Removed the remained tissues and filtrated the protoplast suspension with a 74 um nickel
mesh. Then the suspension was spun at low-speed centrifugation (500 rpm) for 5 min and
washed twice in 0.65 mol/l sucrose. Finally, the protoplasts were resuspended in ddH
2
O
containing 0.65 mol/l sucrose (viscosity 0.01) and divided into two batches. One batch was
immediately subjected to electrophoresis, about at a density of (1.0~2.5)10
5
protoplasts
per ml. The other one was mounted on glass slides and stained with PI for detection of
protoplast quality.
2.3. CELL ELECTROPHORESIS
Cell electrophoresis was carried out according to the method described by Liu et al. (1984)
[22]
. The cell charge was measured using SD-2 cell electrophoresis apparatus, produced by
Shandong University, China. The protoplasts were placed in a rectangular glass, 5cm in
NING Shun-bin et al.
68 Developmental & Reproductive Biology
length and 1.00.5 mm
2
in transverse section area. The salt bridge was 2% agarose gel
containing 0.9% NaCl. The electric field intensity is 10 V/cm. The movements of the
protoplasts were measured in a stationary plane which was determined by the geometry of
the chamber, and confirmed experimentally using instructions provided by the manufacturer
and ensured that there was no drift or electro-osmotic movement of the protoplasts in the
absence of applied electrical potential. The velocity of movement of the protoplasts was
measured by means of a graticule with 90 um markings, placed on the eyepiece. The time
taken by the protoplasts to travel between five markings was measured. Mobility of
individual protoplasts was recorded in both directions by reversal of polarity. 10-15
consecutive runs for each sample were averaged. Each protoplasts sample underwent
electrophoresis for EPM in 15-20 electrophoretic sampling tubes in 7-8 repeated independent
experiments, the averages of electrophoretic display for each sample and the final average of
experiment repeats were taken for statistics. EPM, defined as velocity of protoplasts per unit
electrical field strength, was finally expressed as 10
-3
cm
2
V
-1
S
-1
. Zeta=4EPM
-1
where
is the viscosity of 0.65mol/l sucrose andis dielectric constant of ddH
2
O. This formula
can be reduced to Zeta=140 EPM
[22]
.
3. RESULTS
The method of cell electrophoresis was a sensitive probe for monitoring modifications of the
cell membranes
[23]
. The results of cell electrophoresis for samples taken in different times
indicated that EPM of the protoplasts of root tip cells rose increasingly as the time of
treatment went on. The most obvious effect was shown after treatment with ActD, the next
was that with CHX, and that with COL was shown less obvious change. The EPM values
of the sampling point at 4 hr in former two treatments indicated 20% higher rise than that in
control while that in COL treatment reached the same level as 20% at 6 hr after treatment
(Fig. 1, A).
Fig.1B indicated that the treatment sets of protoplasts with 0.1 mg/l CHX or ActD for 2-8 hr
were suitable for cell electrophoresis (so as in the treatment set of protoplasts with COL for
4-10 hr). The treatment of protoplasts with COL for less than 4-10 hr gave insignificant
difference between treated and control sets in values of EPM. On contrary, when the time
of treatment lasts over 10 hr, obvious decrease in number of protoplasts was shown.
Though it gave significant difference in EPM value, it was not suitable for cell
electrophoresis. It suggested that most of the cells be degraded. So, the criterion for
Electrical Characters on Surface of Apoptotic Cell
Vol. 9 No. 1 (2000) 69
sampling for cell electrophoresis was that the protoplast number should not be obvious
decreased in the treatment with cytotoxins.



















Fig.2 indicated that the preparation of protoplasts for cell electrophoresis was reliable. The
background of protoplast was clear and the cells in both the control and treatment sets were
integral in shape. However, the nuclei of the protoplasts in treatment set were disintegrated
Fig.2 The morphology of the protoplasts and their nuclei in the control and CHX
treatment sets (1000). A: Control; B: Treated with cycloheximide for 8 hr.
Fig.1 A. The changes of EPM in protoplasts after treatments with different cytotoxins were
expressed with Zeta electrical potential (mV). () with ActD; () with CHX; () with COL; and
() control. B. The changes of density of protoplasts after treatment with ActD and COL
respectively as the time of treatment went on.
0
0.5
1
1.5
2
2.5
2 4 6 8 10 12 14h
COL
CHX
c
e
l
l

d
e
n
s
i
t
y
(
1
0
5
/
m
l
)
48
50
52
54
56
58
60
2 4 6 8h
Z
e
t
a

p
o
t
e
n
t
i
a
l

(
m
V
)
A B
NING Shun-bin et al.
70 Developmental & Reproductive Biology
with nuclear fragments scattered in cytoplasm (Fig. 2B), contrary to the control ones, which
were always integral without any destruction (Fig. 2A). The decrease of cell density
indicated in Fig. 1B implied that the treated cells would be disorganized after nuclear
fragmentation. Only a few reports showed that nuclear fragmentation might occur in plant
apoptosis
[6,13]
. However, no morphological difference in cytoplasm was examined; we
couldnt exclude that cytoplasm might condense and then recovered during the
low-infiltration process in the experiment.
4. DISCUSSION
As we known, this is the first experimental evidence of cell electrophoresis in research on
electrical potential of cell membrane in apoptotic cells. Generally, it is believed that many
physical and chemical changes in components of cell membrane like porins and
phospholipids would occur in apoptosis in animals. The degradation of some surface
structures might change surface charge. One of the changes is that the loss of asymmetry of
membrane due to the reverse of phosphate groups which are associated with phospholipids
and contribute to the negative surface charge, from inner to outer surface during apoptosis
process. The second, in early period of apoptosis process, the density of entire membrane
phospholipid is lowered, the membrane structure becomes loose and membrane permeability
increases, which causes the calcium ion current to pour into cytoplasm and sequentially, the
latter will be combined to calmodulin and provokes a series of biochemical reactions. Such
as, the calcium-calmodulin compound can provoke phospholipase A
2
that liberates
unsaturated fatty acid from plasma membrane and forms hydroxyperoxides
[24]
. The third is
that the fusion of endoplasmic reticulum membrane with cell membrane at the late stage of
apoptosis may causes the change of electric charge on cell surface since the structures of the
structures of two organelles is different from each other. Besides, differential expressions
or structures of some membrane proteins in normal and apoptotic cells also cause the
different states of electric charge on cell membrane.
EPM was a measure of electric surface charge determined as a function of different
environmental stimuli
[25]
. These varieties responded for change in surface charge and
displayed different EPM that correlated with the net numbers of charged chemical groups
within surface-exposed parties of their respective at various given time points. Our results
showed surface negative charge had increased during the cytotoxin-induced plant apoptosis
processes (shown in Fig. 2A), suggesting there exists similarity between membrane events of
Electrical Characters on Surface of Apoptotic Cell
Vol. 9 No. 1 (2000) 71
plant cells and apoptosis in animal cells at least in some aspects. In support of this,
exposure of PS to the outer surface of plasma membrane in apoptosis of plant cells triggered
by various agent stimuli
[15,20,21]
and ion leakage and losses of membrane differential
permeability associated with HR cell death
[18,19]
and with senescing wheat petal in daylily
[26]

had been detected. As very accessible is the cell electrophoresis apparatus and the
operation of it, this method might be a novel approach for identification of apoptosis in
combining the observation of morphological changes.
The results from cell electrophoresis also showed a direct correlation between the electric
surface charge of protoplasts and their serious apoptotic features
[16]
and indicated cell
electrophoresis could quantitatively detect the surface change of charge during plant
apoptosis. Cells with a rapid increase of Zeta potential also possessed an early serious
morphological changes and DNA cleavage
[16]
. The method of cell electrophoresis proved
suitable to analyze complex interactions between cells and exogenous factors
[25]
. Further
studies employing chemical modifications and enzymatic treatments on the cells should
prove to be of value in the elucidation of specific ionogenic groups contributing to the
change of charges on the surface of apoptotic plant cells.
The proportion of cellulase and pectinase in mixture for success preparation of protoplasts is
the key element for cell electrophoresis. The appropriate mix proportion of these two
enzymes will be adjusted according to the manufacturers. The criterion for an appropriate
proportion is that, when the cells were dissociated by enzymolysis with pectinase, the
cellulase has to accomplish the digestion of the cell wall and still retain the integrity of the
membrane. Thus, clear isolated protoplasts may be obtained in quantities. In the
meanwhile, the time for enzymolysis must be strictly controlled, otherwise the excessive
enzymolysis will not only destroy protoplast membrane but also destroy interior structure of
cells.





NING Shun-bin et al.
72 Developmental & Reproductive Biology
REFERENCES

1 Caron-Leslie L. A. M.; Evans R. B. and Cidlowski, J. A.: Bcl-2 inhibits glucocorticoid-induced
apoptosis but only partially blocks calcium ionophore or cycloheximide-regulated in S49 cells.
FASEB J., 8:639-645, 1994.
2 Kressel M. and Groscurth P.: Distinction of apoptotic and necrotic cell death by in situ labeling
of fragmented DNA. Cell Tissue Res., 278:549-556, 1994.
3 Lindenboim L.; Haviv R. and Stein R.: Inhibition of drug-induced apoptosis by survival
factors in PC21 cells. J. Neurochem., 64:1054-1063, 1995.
4 Palli S. R.; Sohi S. S.; Cook B. J. et al.: RNA- and protein-synthesis inhibitors induce
apoptosis in a midgut cell line from the spruce budworm, Choristoneura fumiferana. J. Insect.
Physiol., 42: 1061-1069, 1996.
5 Jones B. E.; Lo C. R.; Liu H. et al.: Hepatocytes sensitized to tumor necrosis factor-alpha
cytotoxicity undergo apoptosis through caspase-dependent and caspase- independent
pathways. J. Biol. Chem., 275:705-712 , 2000.
6 Danon A. and Gallois P.: UV-C induces apoptotic-like changes in Arabidopsis thaliana. FEBS
Lett., 437:131-136, 1998.
7 Koukalova B.; Kovarik A.; Fajkus J. et al.: Chromatin fragmentation associated with apoptotic
changes in tobacco cells exposed to cold stress. FEBS Lett., 414:289-292, 1997.
8 Ryerson D. E. and Heath M. C.: Cleavage of nuclear DNA into oligonucleosomal fragments
during cell death induced by fungal infection or by abiotic treatments. Plant Cell., 8:393-402,
1996.
9 Balk J.; Leaver C. J. and McCabe P. F. : Translocation of cytochrome c from the mitochondria
to the cytosol occurs during heat-induced programmed cell death in cucumber plants. FEBS
Lett., 463:151-154, 1999.
10 Katsuhara M.: Apoptosis-like cell death in barley roots under salt stress. Plant Cell Physiol.
38:1091-1093, 1997.
11 Stein J. C.; Hansen G.: Mannose induces an endonuclease responsible for DNA laddering in
plant cells. Plant Physiol., 121:71-80, 1999.
12 Jiang Z. F.; Zhu S.; Sun Y. L. et al.: Induction of apoptosis in purified animal and plant nuclei
by Xenopus egg extracts. Cell Res., 9:79-90, 1999.
13 Wang H.; Li J.; Bostock R. M. et al.: Apoptosis: a functional paradigm for programmed plant
cell death induced by a host-selective phytotoxin and invoked during development. Plant Cell.,

Electrical Characters on Surface of Apoptotic Cell
Vol. 9 No. 1 (2000) 73

8:375-391, 1996.
14 Gilchrist D. G.: Mycotoxins reveal connections between plants and animals in apoptosis and
ceramide signalling. Cell Death Differ., 4:689-698, 1997.
15 O'Brien I. E. W.; Baguley B. C.; Murray, B. G. et al.: Early stages of the apoptotic pathway in
plant cells are reversible. Plant J., 13:803-814, 1998a.
16 Ning S. B.; Song Y. C.; Wang L. et al.: Cytotoxin-induced apoptosis in meristematic cells of
maize roots. Acta Bot. Sin., 43: (in press), 2000.
17 Collins J. A.; Schandi C. A.; Young K. K. et al.: Major DNA fragmentation is a late event in
apoptosis. J. Histochem. Cytochem., 45:923-934, 1997.
18 Mittler R.; Simon L. and Lam E.: Pathogen-induced programmed cell death in tobacco. J.
Cell Sci., 110:1333-1344, 1997.
19 Che F. S.; Iwano M.; Tanaka N. et al.: Biochemical and morphological features of rice cell
death induced by Pseudomonas avenae. Plant Cell Physiol., 40: 1036-1045, 1999.
20 O'Brien I. E. W.; Reutelingsperger C. P. M. and Holdaway K. M.: The use of annexin-V and
TUNEL to monitor the progression of apoptosis in plants. Cytometry. 29:28-33, 1997.
21 O'Brien I. E. W.; Murray B. G.; Baguley B. C. et al.: Major changes in chromatin
condensation suggest the presence of an apoptotic pathway in plant cells. Exp. Cell Res.
241:46-54, 1998b.
22 ; : . .
(). pp126-149, 1986.
23 Sabolovic D.; Canque B.; Berbiquier N. et al.: Stage-dependent alteration of negative charges
of uninfected erythrocytes in Plasmodium falciparum culture. In Vitro Cell Dev. Biol., 27A:
595-596, 1991.
24 Chao M. V.: Ceramide: A potential second messenger in the nervous system. Mol. Cell Sci.,
6:91-96, 1995.
25 Iske U.; Huebner K.; Herold W.: Investigation of the connection between the electrophoretic
mobility (EPM) of microorganisms and their capability of metal uptake. Acta. Biotechnol., 10:
541-549, 1990.
26 Panavas T. and Rubinstein B.: Oxidative events during programmed cell death of daylily
(Hemerocallis hybrid) petals. Plant Sci., 133:125-138, 1998.



NING Shun-bin et al.
74 Developmental & Reproductive Biology






430072

(cell electrophoresis)
(cycloheximide)D (actinomycin D)(colchicine)
(EPM)

Zeta (1A)