Plant Cell Physiol. 37(2): 169-173 (1996)
JSPP © 1996
Salt Stress Induced Nuclear and DNA Degradation in Meristematic Cells of
Barley Roots
Maki Katsuhara and Toshio Kawasaki'
Research Institute for Bioresources, Okayama University, Kurashiki, 710 Japan
Root growth of barley (Hordeum vulgare L., cv.
Akashinriki) was inhibited by 200raMNaCl, when 1 mM
CaCl 2 was present in the hydroponic culture solution. Increasing the CaCl 2 up to 10 mM partially prevented this inhibition. However, inhibition also occurred with 100 mM
NaCl in the presence of 0.1 mM CaCI2. The nuclei of
meristematic cells in roots in which growth had been inhibited by salt stress were studied after staining with DAPI
(4',6-diamino-2-phenyIindol). Nuclear deformation of the
cells occurred with 12 h of salt stress with 500 mM NaCl,
and was followed by degradation. The nuclear degradation
was also observed when the roots were exposed to more
than 300 mM NaCl for 24 h. Biochemical analysis revealed
that nuclear degradation was accompanied by apoptosislike DNA fragmentation. The intracellular mechanisms of
nuclear degradation in cells after salt stress are discussed.
Key words: Apoptosis-like DNA degradation — Barley —
Ca-Na interaction — Root meristematic cells — Salt stress —
Technovit-DAPI method.
Salt stress is one of the most serious problems in agriculture in arid and semi-arid areas. A high concentration of
NaCl greatly reduces growth of both the shoot and the root
(Greenway and Munns 1980, Staples and Tonniessen 1984,
Cheeseman 1988). Unfortunately, the cellular mechanisms
of salt injury in roots are scarcely known. Among the root
cells, meristematic cells are especially interesting, because
mitotic activity and cell division are indispensable for root
growth and because meristematic cells are considered to be
one of the salt-sensitive cells (Huang and Van Steveninck
1988). These cells have been studied under exposure to
moderate salinity (Werker et al. 1983, Huang and Van
Steveninck 1990), but not under more severe salt stress. In
this study, we examined the changes in nuclei of meristematic cells in roots during salt stress by staining the cells
with DAPI (4',6-diamino-2-phenylindol) and observing
them by fluorescent microscopy. We also studied the nuclear DNA for biochemical changes.
Materials and Methods
Plant material—For immersion and sterilization, seeds of
barley (Hordeum vulgare L., cv. Akashinriki) were treated with
0.1% benomyl (Hauptmann et al. 1985) in distilled water for a day
with aeration. Germinated seeds (1 day old) were transplanted and
cultured hydroponically in 3.5 liter pots filled with 0.25 mM
CaSO4 for 2 days and one more day after replacing the medium
with the nutrient solution (4 mM KNO3, 1 mM NaH2PO4> 1 mM
MgSO4, 1 mM CaCl2, 1 mg liter"1 Fe-citrate and pH 5.5 with
NaOH). All the cultures were carried out in the dark in an air-conditioned room (25±0.5°C).
For low- or high-Ca treatment, 0.1 or 10 mM CaCl2 was prepared with the nutrient solution with or without the addition of
NaCl. Before addition of NaCl to nutrient solutions, 10 to 20 seedlings (4 days old) were sampled and root lengths were measured.
Salt stress was carried out by adding NaCl to the nutrient solution. One day (24 h) after salt stress, root lengths of 10 to 20 seedings were measured and root growth for one day was calculated.
The growth of roots without salt stress was calculated as 100% for
every CaCl2 concentration.
Nuclear staining and fluorescent observation—Roots were
separated from shoots and fixed with 20 mM cacodylate plus 4%
formaldehyde (pH 7.4 was adjusted with Tris and the osmolality
with sorbitol to prepare media for salt-stress treatment) for 3 h.
After replacement of water with ethanol, fixed roots were embedded in Technovit 7100 (Kulzer, Germany). This newly developed
resin is useful for fluorescent observation (Kuroiwa et al. 1991).
Samples were sliced 15yum thick, stained with DAPI solution (0.1
^gml" 1 DAPI, 50 mM NaCl, 5 mM EDTA, 10 mM mercaptoethylamine, 10 mM Tris-HCl, pH 7.4) and observed with a fluorescent microscope (Olympus BH2). Stained nuclei showed blue fluorescence with UV excitation.
Nuclei showing deformation or disintegration with remaining
cores were classified as deformed nuclei. Degraded nuclei were defined as those showing disintegration without a clear core. To calculate the percentage of deformed or degraded nuclei, 500 to
1,000 cells in a meristem and 4 to 6 root meristems were examined
for every treatment.
Isolation and analysis of nuclear DNA—Nuclear DNA was
isolated according to Doyle and Doyle (1990). Briefly, about 1 g
fresh weight of root tips (4-6 mm long) were isolated from seedlings and frozen with liq. N 2 . Samples were incubated in buffer
containing 2% hexadecyltrimethylammonium bromide. DNA was
extracted with chloroform-isoamyl alcohol and precipitated with
isopropanol. To remove RNAs, RNase A was used. Isolated DNA
was kept in 0.5 ml buffer (10 mM Tris-HCl (pH 7.4) and 1 mM
EDTA). Samples (10/d) and the marker were subjected to electrophoresis on 2% agarose gel in Tris-acetate EDTA buffer. DNA
was stained with 0.5 ng ml" 1 ethidium bromide for 15 min.
Emeritus professor, Okayama University.
169
170
M. Katsuhara and T. Kawasaki
Results
Fig. 1 presents the effects of NaCl and CaCl2 on root
growth over 24 h. Root growth without NaCl was 27.3,
23.8 and 28.0 mm in 0.1, 1 and 10 mM CaCl2, respectively.
These data suggest that 0.1 mM CaCl2 is enough for the
root growth of 4-day-old seedlings without stress. In 100
mM NaCl, roots treated with 1 and 10 mM CaCl2 grew as
much as in the control. However, root growth was reduced
to 34% in 100 mM NaCl plus 0.1 mM CaCl2. In 200 mM
NaCl, roots with 0.1 and 1 mM CaCl2 showed almost no
growth. On the other hands, partial recovery of the growth
(48%) was observed in roots with 10 mM CaCl2. In more
than 300 mM NaCl, no growth was observed with any of
the CaCl2 treatments.
Fig. 2a shows the DAPI-stained meristem of the control root. Nuclei with smooth and clear boundaries are
observed. After salt stress, the nuclei became deformed
(Fig. 2b) and then degraded (Fig. 2c). Deformed nuclei
appeared 4 h after salt stress (Fig. 3). They increased in
percentage gradually until 12 h after salt stress. Degraded
nuclei were detected 16 h after salt stress and most nuclei degraded within 24 h after salt stress. This degradation was
observed in root cells treated with more than 300 mM NaCl
(Fig. 2d, 4). Increase in external CaCl2 prevented both nuclear deformation caused by 200 mM NaCl (Fig. 5) and nuclear degradation caused by 300 mM NaCl (Fig. 2d, e).
Biochemical analysis revealed that nuclear DNA showed disintegration after salt stress (Fig. 6). Fragmentation of
DNA was clearly detected 8 h after salt stress (Fig. 6A).
The fragmentations were of integral sizes of about 180 (150
to 200) base pairs, but not random. This fragmentation
was considerably suppressed in root cells treated with 500
mM NaCl plus 10 mM CaCl2 for 8 h (Fig. 6B).
Discussion
Increase in the concentration of external NaCl for 24 h
400
NaCl (mol nr')
Fig. 1 Effects of external NaCl and CaCl2 on root growth for 24
h.
A, 0.1 mM CaCl2; O, 1 mM CaCl2; a, 10 mM CaCl2.
Relative growth was calculated after normalizing the root growth
without NaCl as 100%.
inhibited root growth (Fig. 1). When external calcium increased, a higher concentration of NaCl was required to inhibit the growth (Fig. 1). A protective effect of calcium supplementation against salt stress has been reported by many
researchers (Greenway and Munns 1980, Rengel 1992).
Two reasons are possible for the reduced root growth
under salt stress. One is that turgor pressure for cell growth
is lost because of a lower osmotic potential of external media. The other is cell death. To check these hypotheses, the
cells in stressed roots were monitored.
In the meristem, a nucleus occupies most of a cell
(Fig. 2a). Salt stress causes nuclear deformation and subsequent nuclear degradation (Fig. 2b, 2c, 3, 4). When a
nucleus disintegrates, it seems to have lost most of its functions, and cells with such degraded nuclei seem to be dead.
Inhibition of root growth induced by salt stress within 24 h
might be mainly caused by death of the root cells. The
present results do not eliminate the possibility of low external osmotic pressure reducing the turgor of root cells,
resulting in growth inhibition. However, when meristematic cells lose their function due to death, root growth cannot
be recovered even if the turgor is restored by some means.
Nuclear deformation and degradation may be assumed to be caused by dehydration. However, although a new
osmotic equilibrium between cells and external solutions
can be expected to be established within a few minutes after exchange of media, nuclear deformation is hardly detectable 5 min after salt stress (data with shortest time in
Fig. 3). Therefore this deformation was assumed to not be
caused by cellular dehydration. This is supported by the
result that an increase in external CaCl2 prevents nuclear
degradation (Fig. 2d, 2e, 5), because supplemented CaCl2
increases the external osmotic potential rather than decreasing it. When external calcium is low in concentration, salt
stress causes much influx of Na + into cells and disturbs the
cytoplasmic homeostasis (Rengel 1992). Increase in external calcium is proposed to reduce Na + influx into roots and
thus prevent salt injury of cells (Cramer et al. 1989).
Nuclear DNA was isolated from salt-stressed roots
and subjected to electrophoresis (Fig. 6). Isolated DNA consisted of oligonucleosomal fragments (Fig. 6). Such DNA
degradation is one of the characteristics distingishing apoptosis from necrosis in animal cells (Cohen 1993, Martin
et al. 1994). In animali cells, apoptosis are obserbed both
in most programmed cell death and some accidental cell
death (Cohen 1993). In apoptosis, calcium-activated endonuclease cleavages chromatic DNA at the linker site between nucleosomes, resulting in a "ladder structure" of
oligo-nucleosomal fragmentations of sizes in integral units
of 180 base pairs. Since a transient increase in cytoplasmic
calcium is observed in salt-stressed maize cells (Lynch et al.
1989), such an endonuclease can be activated in the cytoplasm of salt-stressed cells. This increase in cytoplasmic calcium is probably driven by disturbance of cytoplasmic
Nuclear degradation in barley root cells
171
Fig. 2 Simultaneous fluorescent and light micrographs of a, meristem of control root; b, meristem of root treated with 500 mM
NaCl+1 mM CaCl2 for 12 h; c, meristem of root treated with 500 mM NaCl + 1 mM CaCl2 for 24 h; d, meristem of root treated with
300 mM NaCl+ 1 mM CaCl2 for 24 h; e, meristem of root treated with 300 mM NaCl+10 mM CaCl2 for 24 h.
Bar: 25 urn.
homeostasis (Rengel 1992). Tilapur et al. (1993) found an
apoptosis-related and calcium-activated endonuclease in
Euglena. This enzyme causes DNA fragmentation in UVilluminated (stressed) Euglena. Mittler and Lam (1995)
found nuclear DNA fragmentation during the differentiation of tracheary elements in pea. They discuss that programmed cell death is thought to occur during the autolysis
of xylem vessels. These findings suggest that cellular mechanism^) like apoptosis may exist in plant cells as in animal
cells.
Structural changes of nuclei caused by salt stress have
been reported by Werker et al. (1983), but they could not
explain the mechanism or physiological meaning of such
phenomenon. In this study, apoptosis-like DNA degrada-
M. Katsuhara and T. Kawasaki
172
O normal nuclei
e deformed nuclei
• degraded nuclei
Fig. 3 Change of nuclei in root meristematic cells after treat
ment with 500 mM NaCl+1 mM CaCl2.
Fig. 6 DNA fragmentation with salt stress.
M, 100 bp-ladder marker. (A) Lane 1, DNA isolated from control root tips; Lane
2, DNA isolated from root tips treated with 500 mM NaCl for 8 h.
(B) Lane 1, DNA isolated from root tips treated with 500 mM
NaCl for 8 h; DNA isolated from root tips treated with 500 mM
NaCl plus 10 mM CaCl2 for 8 h.
O normal nuclei
e deformed nuclei
• degraded nuclei
tion was observed after salt stress. This D N A degradation
should lead to sequential nuclear degradation, cell death
and inhibition of root growth.
100
200
400
300
500
NaCl (mol m-3)
Fig. 4 Effect of salt stress for 24 h on nuclei of cells in root meriCaCl2 concentration was set at 1 mM.
stems.
O normal nuclei
e deformed nuclei
100
QJ
M
CO
u
root
0>
B
80
60
40
e
20
CO
—t
QJ
_)
0
0.1
CaCh
1
10
(mol m-3)
Fig. 5 Effect of calcium during salt stress with 200 mM NaCl for
24 h on nuclei of cells in root meristems.
The authors gratefully thank Dr. M. Shibasaka (Okayama
University) for his discussion and critical reading of the manuscript. The authors wish also to thank Dr. T. Kuroiwa (University
of Tokyo) for his kind advice and many suggestions about
Technovit-DAPI methods.
References
Cheeseman, J.M. (1988) Mechanisms of salinity tolerance in plants. Plant
Physiol. 87: 547-550.
Cohen, J.J. (1993) Apoptosis. Immunology Today 14: 126-130.
Cramer, O., Epstein, E. and Lauchli, A. (1989) Na-Ca interactions in
barley seedlings: relationship to ion transport and growth. Plant Cell Environ. 12: 551-558.
Doyle, J.J. and Doyle, J.L. (1990) Isolation of plant DNA from fresh
tissue. Focus 12: 13-15.
Greenway, H. and Munns, R. (1980) Mechanisms of salt tolerance in
nonhalophytes. Annu. Rev. Plant Physiol. 31: 149-190.
Hauptmann, R.M., Widholm, J.M. and Paxton, J.D. (1985) Benomyl: a
broad spectrum fungicide for use in plant cell and protoplast culture.
Plant Ceil Rep. 4: 129-132.
Huang, C.X. and Van Steveninck, R.F.M. (1988) Effect of moderate salinity on patterns of potassium, sodium and chloride accumulation in cells
near the root tip of barley: role of differentiating metaxylem vessels.
Physiol. Plant. 73: 525-533.
Huang, C.X. and Van Steveninck, R.F.M. (1990) Salinity induced structural changes in meristematic cells of barley roots. New Phytol. 115: 1722.
Kuroiwa, T., Fujie, M., Mita, T. and Kuroiwa, H. (1991) Application of
embedding of samples in Technovit 7100 resin to observations of small
amounts of DNA in the cellular organelles associated with cytoplasmic
inheritance. Appl. Fluorescence Techno/. 3: 23-25.
Nuclear degradation in barley root cells
Lynch, J., Polito, V.S. and Lauchli, A. (1989) Salinity stress increases cytoplasmic Ca activity in maize root protoplasts. Plant Physiol. 90: 12711274.
Martin, S.J., Green, D.R. and Cotter, T.G. (1994) Dicing with death:
dissecting the components of the apoptosis machinery. TIBS 19: 26-30.
Mittler, R. and Lam, E. (1995) In situ detection of nDNA fragmentation
during the differentiation of tracheary elements in high plants. Plant Cell
Physiol. 108: 489-493.
Rengel, Z. (1992) The role of calcium in salt toxicity. Plant Cell Environ.
15: 625-632.
173
Staples, R.C. and Tonniessen, G.H. (1984) Salinity Tolerance in Plants p.
443. Jon Wiley and Sons, New York.
Tilapur, U.K., Treml, S., Thar, B., Hader, D.-P. and Scheuerlein, R.E.
(1993) UV-B induced DNA fragmentation in Euglena gracilis is mediated
by an influx of Ca 2+ and activation of a Ca2+-dependent nuclease. In
Abstracts of XV International Botanical Congress, pp. 473. Yokohama.
Werker, E., Lerner, H.R., Weimberg, R. and Poljakoff-Mayber, A. (1983)
Structural changes occurring in nuclei of barley root cells in response to
a combined effect of salinity and ageing. Amer. J. Bot. 70: 222-225.
(Received June 22, 1995; Accepted December 28, 1995)