全 文 ：Received 5 Jan. 2004 Accepted 10 Apr. 2004
Supported by the National Natural Science Foundation of China (39970072).
* Author for correspondence. Tel: +86 (0)10 62772678. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (6): 711-718
Increase in Methylene Resonance Signal Intensity Is Associated
with Apoptosis in Plant Cells as Detected by 1H-NMR
ZHANG Gui-You1, ZHU Rui-Yu1, XU Ye1, YAN Yong-Bin1, 2, DAI Yao-Ren1*
(1. Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China;
2. State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing 100084, China)
Abstract: Apoptosis, both in animal and plant cells, is usually analyzed by observing and detecting the
characteristic morphological and biochemical changes in nuclei, organelles such as mitochondria and cell
membranes. Using 1H-NMR we have found a correlation between the progression of apoptosis and 1H-NMR
methylene signal intensity in cultured tobacco (Nicotiana tabacum L. cv. BY-2) and carrot (Daucus carota
L.) cells. The increase in the methylene resonance signal intensity which reflects the alterations in
membrane lipids and accordingly in the biophysical and biochemical characteristics of cell membrane
during apoptosis was found to be closely associated with nuclear changes as detected by DNA laddering
assay and the TUNEL procedure. The rise of methylene resonance signal intensity was evident in apoptotic
plant cells induced by various factors including nicotinamide, menadione, heat shock and hydroxyl radicals,
but it was not found in necrotic cells. To our knowledge, this is the first report of the 1H-NMR detection of
apoptosis which is closely associated with the increase in methylene resonance signal intensity in plants.
Key words: apoptosis; plant cells; 1H-NMR; membrane lipids
Apoptosis is an important physiological process lead-
ing to the elimination of redundant or harmful cells during
the development of organisms. In animal cells apoptosis is
characterized by a series of typical morphological events,
such as shrinkage of the cells, membrane blebbing, and
nuclear condensation. The activation of endonuclease and
translocation of phosphatidylserine have been discovered
as biochemical hallmarks in cells undergoing apoptosis
(Wyllie,1980). The final stage of apoptosis is the fragmen-
tation of the cell into “apoptotic bodies” that are phago-
cytosed by other cells (Hengartner, 2000). In plants,
apoptosis is known to also play an important role in devel-
opment and defense mechanisms, such as cell death dur-
ing xylogenesis, aerenchyma formation, leaf senescence
and reproductive processes (Pennel and Lamb, 1997).
Furthermore, apoptosis was found to be involved in the
hypersensitive response of plants to pathogens or in re-
sponse to a variety of abiotic factors (Greenberg et al.,
1994; Wang et al., 1996). A number of morphological and
biochemical similarities were found between animal and
plant cells undergoing apoptosis (de Jong et al., 2000).
Increasing evidence indicates that plant and animal cells
share some common pathways while undergoing apoptotic
cell death.
In animal cells it has been reported that during
apoptosis profound alterations take place in the cell
membrane, including changes in lipid packing, membrane
microviscosity and loss of membrane asymmetry (Fadok
et al., 1992; Homburg et al., 1995). More recently
Blankenberg and his coworkers reported that apoptosis
was associated with at least a twofold rise in the amount of
methylene protons observable by 1H-NMR in T- and B-
cell lymphoblasts, myeloblast, Hela cells, and 3T3 fibro-
blast (Blankenberg et al., 1996; 1997). On the other hand,
there are also reports of alterations in membrane lipids in
plant cells in response to stress (Welti et al., 2002) and
while undergoing apoptosis (O’Brien et al., 1997). Our pre-
vious study also found a loss of membrane asymmetry
with the surface exposure of membrane phosphatidylserine
as detected by Annexin-V assay (Lei et al., 2003). The aim
of this study is to further reveal the changes of membrane
lipids during apoptosis in plant cells using 1H-NMR
spectroscopy. We have observed remarkable increases in
methylene signal intensity in apoptotic tobacco cells in-
duced by nicotinamide, menadione, heat shock, hydroxyl
radicals and in carrot cells treated with nicotinamide. The
rise of methylene resonance signal intensity which mirrors
alterations in the cell membrane in apoptotic tobacco cells
started 9 h after nicotinamide treatment and coincided with
the onset of internucleosomal DNA fragmentation. Our
results suggested that 1H-NMR is useful for apoptosis
detection in plant cells and that the increase in methylene
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004712
resonance signal intensity is a common event which takes
place in apoptosis of both animal and plant cells.
1 Materials and Methods
1.1 Cell culture
Tobacco (Nicotiana tobacum L. cv. BY-2) and carrot
(Daucus carota L.) suspension cells were cultured in MS
medium supplemented with 0.6 mg/L and 1.0 mg/L 2,4-D
(2,4-dichlorophenoxyacetic acid) at 26 ºC under rotation
(120 r/min). Suspension cells were subcultured every five
days. Exponentially growing cells were used for this study.
1.2 Treatment of tobacco and carrot suspension cells by
chemical inducers
Stock solutions of chemicals were added to flasks con-
taining either tobacco or carrot suspension cells until the
anticipated concentrations were reached, and cells were
incubated under normal culture conditions at 26 ºC for 24 h
at 120 r/min rotation. The final concentration of chemicals
used to induce apoptosis was 250 mmol/L for nicotinamide,
300 mmol/L for menadione, and 2.0 mmol/L FeSO4/1.0
mmol/L H2O2 for hydroxyl radical generation.
1.3 Heat shock treatment
Flasks containing tobacco cells were immersed in a wa-
ter bath at 44 ºC under rotation of 120 r/min for 4 h. After
heat treatment, cells were returned to the normal culture
conditions at 26 ºC for 20 h for recovery. The necrotic cells
were obtained by incubating tobacco cells at 60 ºC for 0.5
h.
1.4 Test of percentage of apoptosis
Cel ls were s ta ined wi th TUNEL (Termina l
deoxynucleotidyltransferase-mediated dUTP nick end
labeling) reagent and 4,6-diamidino-2-phenyl-indole
(DAPI), respectively. Cells with nuclei that could be stained
by the TUNEL reagent were regarded as apoptotic. The
total cell number was counted based on DAPI staining,
and the ratio of cells stained by TUNEL to the total cells
was calculated as the percentage of apoptosis.
1.5 DNA laddering assay
Tobacco DNA electrophoresis was conducted accord-
ing to Mittler and Lam (1995). In brief, cells were frozen in
liquid nitrogen and ground to a fine powder. Then an ex-
traction buffer (200 mmol/L Tris-HCl, pH 7.5, 25 mmol/L
EDTA, 250 mmol/L NaCl, 0.5% sodium dodecyl sulfate)
was added. After phenol/chloroform extraction followed
by ethanol precipitation, DNA was transferred to a TE buffer
(10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0) and RNase diges-
tion was then conducted. DNA was loaded on a 1.8% aga-
rose gel and run at 5 V/cm for 2 h. DNA fragments were
then visualized by ethidium bromide staining.
1.6 In situ detection of DNA fragmentation by the TUNEL
procedure
The TUNEL procedure was conducted according to
the manufacturer’s instructions (Boehringer Mannheim,
Germany). Cells were fixed with 4% paraformaldehyde (in
PBS, pH 7.4) for 30 min at room temperature, and then incu-
bated in permeabilization solution (0.1% Triton X-100 in
0.1% sodium citrate) for 2 min on ice. After the cells were
rinsed twice with PBS (pH 7.4), 50 mL treated suspension
cells were drawn out and mixed with 20 mL TUNEL reaction
mixture and incubated for 1 h at 37 ºC. Finally, cells were
rinsed with PBS and observed under a fluorescence micro-
scope (Nikon E-600 10×20).
1.7 1H-NMR analysis
Intact cells were counted, washed twice with PBS-H2O,
and twice with PBS-D2O. Cells were stored at 4 ºC before
the NMR experiments. Cells were resuspended in 500 mL
(about 4×106 cells/mL) of PBS-D2O and then were trans-
ferred into a 5-mm NMR tube. All 1H-NMR experiments
were performed on a Varian Unity Inova 500NB
spectrometer, located at the Department of Biological Sci-
ences and Biotechnology, Tsinghua University. The NMR
sample was stabilized at room temperature for 5 min and
then inserted into the magnet preequilibrated to a tem-
perature of 25 ºC. The 1H-NMR spectrum was recorded
after 15 min equilibrium, with 64 transients and a spectral
width of 6 000.6 Hz. The improved WATERGATE pulse
sequence (Liu et al., 1998) was used for water suppression
in all experiments. All data were processed and analyzed
using the VNMR or WinNuts software provided by Varian
(Palo Alto, CA). No window function was used before Fou-
rier transformation and the obtained spectra were calibrated
to the residual water signal at 4.7 ppm. Signal assignments
Fig.1. Time course of apoptosis induced by 250 mmol/L nico-
tinamide in tobacco suspension cells. Tobacco suspension cells
sampled at different treatment time were stained with 5 mg/mL
DAPI. Cells that contained nuclei showing chromatin condensa-
tion and nuclear collapse were counted as apoptotic cells.
ZHANG Gui-You et al.: Increase in Methylene Resonance Signal Intensity Is Associated with Apoptosis in Plant Cells as
Detected by 1H-NMR 713
were referenced to previous works (Mountford et al., 1982;
Mountford and Tattersall, 1987).
2 Results
2.1 Apoptosis induced by nicotinamide in tobacco cells
and the 1H-NMR spectra of apoptotic cells
To determine if there is a relationship between apoptosis
and methylene resonance signal intensity which reflects
lipid-related alterations in cell membrane, we studied the
time course of spectral changes in apoptotic tobacco cells
induced by nicotinamide. In our previous studies we found
that 250 mmol/L was an effective concentration for nicoti-
namide to induce apoptosis (Zhang et al., 2003). The per-
centage of apoptosis was monitored by the TUNEL proce-
dure (Figs.1, 2). It was found that apoptosis in tobacco
cells induced by nicotinamide increased steadily with time.
The percentage of apoptosis reached 43% at 9 h and 95%
at 24 h after the addition of nicotinamide. As shown in
Fig.3, internucleosomal DNA fragmentation was first ob-
served at 9 h and peaked at 24 h after nicotinamide treatment.
Fig.2. In situ detection of DNA cleavage in tobacco suspension cells. a, b. Control cells stained by DAPI and TUNEL, respectively.
c, d. Cells treated by 250 mmol/L nicotinamide for 9 h and stained by DAPI and TUNEL, respectively. e, f. Cells treated by 250
mmol/L nicotinamide for 24 h and stained by DAPI and TUNEL, respectively.
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004714
Strikingly, the signal intensity of methylene resonance
arose distinctly showing a similar time course with a two-
fold rise in methylene resonance signal intensity at 24 h
after nicotinamide addition (Figs.4, 5). On the other hand,
the intensity of choline resonance signal intensity de-
creased with time, displaying opposite kinetics. These re-
sults indicated a distinct correlation between apoptosis
progression and the changes in methylene and choline
resonance signal intensity in tobacco cells.
2.2 1H-NMR spectra of tobacco cells treated with various
apoptosis inducers
In our previous studies we have established that heat
shock, menadione and hydroxyl radicals are efficient
apoptosis inducers in plant cells (Chen et al., 1999; Xia et
al., 1999; Sun et al., 2000; Lei et al., 2003). Using these
physical and chemical inducers at appropriate doses, to-
bacco cells were induced to undergo apoptosis as detected
by the TUNEL procedure. 1H-NMR spectra of these cells
displayed a significant increase in methylene resonance
signal intensity characteristic to apoptotic cells (Fig.6).
The results suggested that the rise of methylene resonance
signal intensity is a common event of apoptosis in tobacco
cells. This was further confirmed by comparison of 1H-
NMR spectra of apoptotic and necrotic cells. When to-
bacco cells were incubated at 60 ºC for 30 min non-
apoptotic necrotic cell death was observed by microscopic
observations and using the TUNEL assay to verify the
observations (data not shown). No typical increase in
methylene resonance signal intensity was seen for necrotic
cells. Furthermore, the decrease in choline resonance sig-
nal intensity which was shown in nicotinamide-induced
tobacco cells was no longer observable in apoptotic cells
induced by other inducers (Fig.6).
2.3 1H-NMR spectral changes in apoptotic carrot cells
To find out if the rise of methylene resonance signal
intensity is a general property of apoptosis in plant cells,
we also studied the 1H-NMR spectrum of carrot cells. When
carrot cells were treated with 250 mmol/L nicotinamide,
80% of the cells were found to be apoptotic as detected by
TUNEL assay, and a 1.5-fold increase in methylene reso-
nance signal intensity was observed. At the same time, a
more dramatic decline in choline resonance signal inten-
sity was shown in these apoptotic carrot cells (Fig.7).
In sum, the evidence from our study indicated that the
increase in methylene resonance signal intensity which
mirrors lipid-related profound alterations in cell membrane
may be considered as a general hallmark of apoptosis in
Fig.3. Apoptosis in tobacco suspension cells. DNA fragmen-
tation in tobacco cells treated by 250 mmol/L nicotinamide. Lane
M, DNA molecular markers; Lanes 1-7, DNA extracted at 0, 3,
6, 9, 15, 18, and 24 h after treatment.
Fig.4. The observations of 1H-NMR spectra recorded
continously at different time points for tobacco suspension cells
treated with 250 mmol/L nicotinamide. The choline protons at
3.2 ppm, methylene protons at 1.3 ppm , and methyl protons at
0.9 ppm.
ZHANG Gui-You et al.: Increase in Methylene Resonance Signal Intensity Is Associated with Apoptosis in Plant Cells as
Detected by 1H-NMR 715
plant cells. Our finding is consistent with the 1H-NMR
spectral changes in apoptotic animal cells reported by
Blankenberg and his coworkers and suggested that the
rise in methylene resonance signal intensity may be a com-
mon characteristic of apoptotic animal and plant cells.
3 Discussion
Apoptotic cells display characteristic alterations in vari-
ous cellular compartments including cell membrane.
Apoptosis has been found to be associated with profound
biophysical and biochemical changes in cell membrane
such as lipid packing in the lipid bilayer, membrane
microviscosity and loss of membrane asymmetry with the
surface exposure of membrane components. These
changes result in the increase in methylene resonance sig-
nal intensity (Steller, 1995; Thompson, 1995). Blankenberg
et al. (1996) provided convincing evidence for the asso-
ciation of the onset of apoptosis with the rise of methyl-
ene resonance signal intensity in different animal cells.
Our study, on the other hand, for the first time revealed the
same relationship between apoptosis and the changes of
methylene resonance signal intensity in plant cells treated
by various apoptosis-inducing agents, both physical and
chemical. Our results thus suggest that a common mecha-
nism of apoptosis is shared by both animal and plant cells.
Although the decrease of choline resonance which at
least partially reflects degradation of phosphocholine in
membrane, may also be related to apoptosis to a certain
extent in nicotinamide-treated tobacco and carrot cells, in
cells treated by other apoptosis-inducers this relationship
is no longer observable.
Moreover, in apoptotic tobacco cells the increase in
methylene resonance signal intensity was found to be syn-
chronized with but not earlier than internucleosomal DNA
fragmentation as reported in apoptotic animal cells. The
cause of the difference is unclear at the present time.
The plasma membrane undergoes multiple changes dur-
ing apoptosis (Morris et al., 1984), which reflect the differ-
ences between normal cells and those undergoing
apoptosis (Engeland et al., 1997). Therefore, many meth-
ods has been developed to detect apoptosis according to
the membrane alterations (O’Brien et al., 1997; Woodle
and Kulkarni, 1998; Wang et al., 2002). We can distinguish
Fig.5. The ratio curve of CH2/CH3 and choline/CH3. The
relative areas underneath the CH2 and CH3 resonances were cal-
culated by the method of integration of the proton spectrum,
and the trough between the CH2 and CH3 was used as baseline
reference.
Fig.6. 1H-NMR spectra recorded from treated tobacco sus-
pension cells. a. Control cells. b. Cells treated at 60 ºC for 0.5 h
(necrosis). c. Cells treated with 2.0 mmol/L FeSO4/1.0 mmol/L
H2O2 for 48 h. d. Cells treated with 300 mmol/L menadione for
48 h. e. Cells treated at 44 ºC for 4 h and then returned to the
normal culture conditions at 26 ºC for 20 h for recovery.
Fig.7. 1H-NMR spectra recorded from treated carrot suspen-
sion cells. a. Control cells. b. Cells treated with 250 mmol/L
nicotinamide at 26 ºC for 24 h.
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004716
dead cells from viable cells by staining based on trypan
blue exclusion. This is a classical standard method with
the advantages of being quick and cheap, but it is not
suitable for detection of apoptosis. FITC-annexin V has
been shown to provide a sensitive probe for detecting
apop tos is . T his re f lec ts t he t rans loca t io n o f
phosphatidylserine from the inner to the outer leaflet of
the plasma membrane during apoptosis (Koopman et al.,
1994). However, annexin V can also stain the inner mem-
brane of ruptured cells. Thus, apoptotic cells must be dis-
tinguished from necrotic cells with an additional DNA stain.
Because a loss of mitochondrial membrane potential ( DYm)
occurs during apoptosis, analysis of DYm is also used to
distinguish apoptotic cells. But the plasma membrane might
mimic alteration of DYm in some circumstances (Salvioli et
al., 1997). In recent years, a new method has been reported
to detect apoptosis in HL-60 cells (Wang et al., 2002). This
method is based on the specific membrane capacitance
and conductivity of cells during apoptosis. This method
can be used to detect dielectric changes at least an hour
before the increase of phosphatidylserine could be
detected, so it provides information about apoptosis in
the electrochemistry alteration of plasma membrane. More
importantly, this method does not alter cell viability. An-
other noninvasive and rapid method to detect apoptosis
is proton nuclear magnetic resonance spectroscopy
(Blankenberg et al., 1996), which is based on the alter-
ations of membrane phospholipids and microviscosity.
The CH2/CH3 signal intensity ratio is coincident with the
increases in annexin V-positive cells during apoptosis, and
the ratio can be used to estimate the percentage of
apoptotic leukemic cells in vivo (Blankenberg et al., 1997).
A similar result was reported in A549/DDP cells (Huang et
al., 2003).
The most commonly used methods for detecting
apoptosis at the present time are based on the nuclear
alterations during apoptosis. The application of proton
nuclear magnetic resonance spectroscopy in apoptosis
detection provided an alternative and precise assay based
on the alterations in cell membrane. In addition to its po-
tential in clinical use and ability to provide noninvasive
detection of apoptosis, the 1H-NMR study on apoptosis
will also be helpful in revealing the fine mechanisms of
apoptotic cell death.
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