Annals of Botany 86: 983±994, 2000
doi:10.1006/anbo.2000.1260, available online at http://www.idealibrary.com on
E€ect of Di€erent Gravity Environments on DNA Fragmentation and Cell Death in
KalanchoeÈ Leaves
M . C . P E D RO S O * { and D. J . D UR Z A N{
{Dept. Biologia Vegetal, Faculdade de CieÃncias de Lisboa, Ed. C2, Piso 1, Campo Grande, 1749-016 Lisboa, Portugal
and {Dept Environmental Horticulture, University of California, Davis, CA 95616-8587, USA
Received: 19 May 2000 Returned for revision: 19 June 2000 Accepted: 12 July 2000
Di€erent gravity environments have been shown to signi®cantly a€ect leaf-plantlet formation and asexual
reproduction in KalanchoeÈ daigremontiana Ham. and Perr. In the present work, we investigated the e€ect of gravity
at tissue and cell levels. Leaves and leaf-plantlets were cultured for di€erent periods of time (min to 15 d) in di€erent
levels of gravity stimulation: simulated hypogravity (1 rpm clinostats; 2 10 ÿ4 g), 1 g (control) and hypergravity
(centrifugation; 20 and 150 g). Both simulated hypogravity and hypergravity a€ected cell death (apoptosis) in this
species, and variations in the number of cells showing DNA fragmentation directly correlated with nitric oxide (NO)
formation. Apoptosis in leaves was more common as gravity increased. Apoptotic cells were localized in the epidermis,
mainly guard cells, in leaf parenchyma, and in tracheary elements undergoing terminal di€erentiation. Exposures to
acute hypergravity (up to 60 min) showed that chloroplast DNA fragmentation occurred prior to nuclear DNA
fragmentation, marginalization of chromatin, nuclear condensation, and nuclear blebbing. Addition of sodium
nitroprusside (NO donor) mimicked centrifugation. NO and DNA fragmentation decreased with NG-monomethyl-Larginine (NO-synthase inhibitor). The variations in NO levels, nucleoid DNA fragmentation, and cell death show how
# 2000 Annals of Botany Company
chloroplasts, cells and leaves may respond (and adapt) to gravity changes.
Key words: Apoptosis, chloroplasts, gravitational biology, KalanchoeÈ daigremontiana, mechanical stress, Mother of
Thousands, nitric oxide, programmed cell death, TUNEL.
I N T RO D U C T I O N
Traumatic (`accidental') cell death, often called necrosis, is
a non-physiological process caused by overwhelming levels
of stresses (e.g. toxins, heat or cold), which involves
disruption of membrane integrity and subsequent cellular
swelling and lysis ( for a review see Schwartzman and
Cidlowski, 1993; Gray and Johal, 1998). Programmed cell
death (PCD) is a physiological cell death process, dynamically regulated by both developmental and environmental
cues, involving the selective elimination of unwanted cells
( for a review see Ellis et al., 1991; Havel and Durzan,
1996b; Jacobsen et al., 1997; Pennel and Lamb, 1997;
Gilchrist, 1998; Gray and Johal, 1998; Yeng and Yang,
1998). When PCD involves the marginalization of chromatin in the nucleus, nuclear condensation, shrinkage of the
cytoplasm, cleavage of nuclear DNA into oligonucleosomes
(Ryerson and Heath, 1996), and the breakage of the
cytoplasm or nucleus into small, sealed packets, the process
is termed apoptosis, both in animals (Kerr et al., 1972) and
plants (Havel and Durzan, 1996a; Wang et al., 1996; Yeng
and Yang, 1998). The in situ detection of DNA fragmentation leading to cell death can be achieved by the terminal
deoxynucleotidyl transferase-mediated dUTP nick end
labelling of DNA 30 -OH groups (TUNEL method)
(Gavrieli et al., 1992; Wang et al., 1996).
* For correspondence at: Dept Environmental Horticulture, University of California, Davis, CA 95616-8587, USA. Fax ‡530 752
1819, e-mail [email protected]
0305-7364/00/110983+12 $35.00/00
In plants, the continuous generation of organ systems by
the plant's meristems is countered by the programmed
senescence and/or abscission (shedding) of existing organs
throughout the life of the plant (Bleecker and Patterson,
1997). Senescence involves cell death on a large scale,
nutrient recycling and is often interrelated with reproduction ( for reviews see Resende, 1951; Barlow, 1982; Bleecker
and Patterson, 1997; Pennel and Lamb, 1997; Gray and
Johal, 1998; NoodeÂn, 1988). Recent reports have shown
that the process of senescence exhibits features of PCD by
apoptosis (in Gray and Johal, 1998; Yeng and Yang, 1998).
KalanchoeÈ daigremontiana Ham and Perr. (Crassulaceae),
commonly known as Mother of Thousands, reproduces
asexually by forming plantlets in leaf indentations (Warden,
1970a, b). In nature, its entire plant body except leafplantlets senesces as a consequence of ¯oral di€erentiation
(Resende, 1951). The leaf-plantlets fall to earth and convert
into adult plants. At unit gravity, in nature and in vitro,
PCD precedes plantlet detachment from the mother-leaf,
leading to an abscission scar after plantlet fall (Pedroso,
1998). Recent work showed that changes in gravity
signi®cantly a€ected leaf-plantlet formation, development,
and asexual reproduction in this species (Pedroso and
Durzan, 1998). The cytological, morphological and physiological changes observed suggested that stressful gravity
environments (hypergravity) interfered with cellular processes in ways that might trigger apoptosis.
The response of K. daigremontiana in natural conditions
(Resende, 1951) and in hypergravity (Pedroso and Durzan,
# 2000 Annals of Botany Company
984
Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis
1998, 1999) suggested that, under stressful conditions, leaf
senescence could be induced and used as a mechanism for
plant survival. As a ®rst step to verify this hypothesis, we
investigated the e€ect of gravity (simulated hypogravity,
1 g, and hypergravity) on cell death in KalanchoeÈ leaves.
Nitric oxide (NO) is a free radical which plays an
important role as an intra- and intercellular messenger in
animal systems ( for reviews see Lancaster, 1992; Kojima
et al., 1998). In plants, NO formation is associated with
ethylene biosynthesis (Leshem and Haramaty, 1996),
disease resistance (Delledonne et al., 1998; Durner et al.,
1998), drought stress (Leshem, 1996; Haramaty and
Leshem, 1997), cell death and cell proliferation (Magalhaes
et al., 1999; Pedroso et al., 1999, 2000a, b). Evidence for its
function as an endogenous maturation and senescence
regulating factor was recently provided (Leshem et al.,
1998). Histochemical detection of NO in vivo can now be
achieved using the probe 4,5-diamino¯uorescein diacetate
(DAF-2 DA) (Kojima et al., 1998; Magalhaes et al., 1999;
Pedroso et al., 2000a, b). The potential involvement of NO
as trigger of cell death in KalanchoeÈ leaves was also
investigated in the present work.
Drastic hypergravity treatments, for short (15 d) and
acute (10±60 min) periods of time, were chosen to ascertain
the induction of large-scale cell death ( for a review see Gray
and Johal, 1998), helping the cytological distinction
between physiological (PCD) and non-physiological (`accidental') cell death. Double staining TUNEL-DAPI, microscopy techniques, a NO probe (DAF-2 DA), a NO donor
(sodium nitroprusside, SNP) and a competitive substrate
inhibitor (NG-monomethyl-L-arginine, L-NMMA) of the
NO-forming enzyme NO-synthase (EC 1.14.13.39; NOsynthase), were used to show that leaf and leaf-plantlet
exposure to di€erent gravity environments a€ects leaf PCD
in this species, and that variations in the number of cells
showing DNA damage in chloroplasts and nuclei is directly
correlated with NO formation.
M AT E R I A L S A N D M E T H O D S
Plant material and in vitro culture conditions
KalanchoeÈ daigremontiana Ham. and Perr. shoot cultures
were used as a source of leaves and leaf-plantlets (Pedroso,
1998). Stock shoot cultures were maintained by micropropagation on half-strength Murashige and Skoog basal
medium (1962), with 5 mg l ÿ1 dithiothreitol, 25 g l ÿ1
D-glucose, without glycine and growth regulators (MS/2
medium); pH was adjusted to 5.5 prior to agar addition
(7.5 g l ÿ1) and medium sterilization (10 ÿ5 Pa, 1208C, for
20 min). Subculture was performed every 4 weeks. Unless
otherwise stated, cultures were maintained in glass tubes
(150 22 mm), at 258C, under a 16 h photoperiod
provided by Phillips F40 AGRO AGROLITE ¯uorescent
lamps [maximum radiation at 350±450 nm (blue) and
650 nm (red)]. Leaves and leaf-plantlets were cultured on
solid MS/2 medium, on plastic Petri dishes (90 mm) sealed
with transparent ®lm. Leaf-plantlets were cultured in a
vertical position with their roots in the medium. Leaves
were inoculated horizontally with the upper leaf-page up.
Cultures under hypergravity were kept in darkness, whereas
those exposed to simulated hypogravity were kept in
darkness or under a 16 h photoperiod. Leaf-plantlets and
leaves cultured under the Earth's gravity (1 g), in darkness
and under a 16 h photoperiod, were used as controls.
Hypergravity and simulated hypogravity experiments
Low speed centrifuges (Braunn Knecht-Heimann, Co.,
San Francisco, International Equipment Company, Boston,
MA, USA) with wood carriers were used to simulate
hypergravity. The centrifuges were modi®ed to prevent
temperature oscillations and vibration. Leaves and leafplantlets were exposed to 20 and 150 g, for 15 d, or to 150 g
for 10, 30 and 60 min. Clinostats equipped with 1 rpm
motors and adapted to hold a Modular Incubator Chamber
(Billups-Rotenberg, Inc., Burlingame, CA, USA) were used
to simulate hypogravity (2 10ÿ4 g). Leaves and leafplantlets were clinorotated, in darkness and under a 16 h
photoperiod, for 15 d. Immediately after each treatment,
samples (leaves) were collected and immersed for 24 h, at
48C, in 3.7 % formaldehyde ®xative. Ninety leaf-plantlets
and 30 leaves were cultured in each experiment. Experiments were repeated four times. In both hypergravity and
hypogravity experiments, no ethylene production was
detected, nor were oxygen, CO2 , temperature and vibration
limiting factors (Pedroso, 1998, 1999).
In situ detection of DNA fragmentation
Nucleoid and nuclei DNA fragmentation in leaf cells was
detected in situ using the terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP nick end labelling
(TUNEL) method (Pedroso, 1998, 1999). Brie¯y, ®xed
leaves were hand-sectioned with a razor blade and the
sections washed in PBS (10 mM NaH2PO4 , 150 mM sodium
chloride, pH 7.2), ®ve times over 1 h, at room temperature
(RT). The sections were then sequentially immersed in an
enzymatic solution [4 % (v/v) pectinase (Sigma Chemical
CO, St Louis, MO, USA) and 2 % (w/v) Cellulase (Fluka
Biochemika, CH) in PBS (30 min, at RT); PBS (5 min at
RT); methanol in 1 % acetic acid (20 min); PBS (5 min at
RT), and incubated with 10 mg ml ÿ1 proteinase K (Boehringer Mannheim, Germany) in PBS, pH 7.5, for 15 min at
378C]. Following the proteinase treatment, the sections
were rinsed in PBS for 2 min and immersed in TDT bu€er
(30 mM Trisma base, 140 mM sodium cacodylate, 1 mM
cobalt chloride, pH 7.2), for 2 min, at 378C. The sections
were then incubated for 60 min at 378C in the TUNEL
reaction mixture (Boehringer Mannheim, Germany) consisting of 45 ml of TUNEL label ( ¯uorescein-dUTP and
dNTP) and 5 ml of TUNEL enzyme (terminal deoxynucleotidyl transferase). After incubation, the sections were
washed in TB bu€er (300 mM NaCl, 30 mM sodium citrate,
pH 8.5) for 15 min, at 378C, to terminate the reaction, and
®nally rinsed in PBS, at RT. Controls were performed for
each step of the protocol to determine potential sources of
artifacts, namely by the use of enzymes and of methanol in
acetic acid. Results showed that incubation in the enzymatic
mixture is not required, it has to be optimized for each plant
Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis
material, and should be avoided if cells or thin sections are
used. Also, higher proteinase K concentrations can lead to
false TUNEL-positive nuclei. For negative controls, only
TUNEL label was added. Incubation of ®xed sections with
DNase-I (1 mg ml ÿ1, 10 min, at RT) prior to TUNEL
labelling was performed for positive controls. Counterstaining for 15 min, at RT, with 1 mg ml ÿ1 40 -6-diamino-2phenylindole dihydrochloride (DAPI), which speci®cally
binds to adenine-thymine-rich deoxyribonucleic acids, was
performed to distinguish non-apoptotic nuclei from
apoptotic ones. Sections were mounted in Vectashield
(Vector Laboratories Inc., Burlingame, CA, USA) and
observed by ¯uorescence and/or confocal laser scanning
microscopy (CLSM). TUNEL-positive nuclei or nucleoids
¯uoresce green (excitation 450±500 nm, detection 515±
565 nm), while DAPI-positive nuclei ¯uoresce blue (emission 365 nm, excitation 510±530 nm). Four leaves per
treatment were randomly collected from each of the four
experiments performed (see above) and used in TUNEL
assays. DAPI and TUNEL-positive cells were counted in
ten individual ®elds from at least six leaf sections per leaf.
Experiments reported herein were repeated at least four
times, with all results in agreement between experiments.
Results are presented as mean values + s.d. Simple analysis
of variance (ANOVA) was performed for P 5 0.05 (results
not shown).
Visualization of nitric oxide
Fresh leaves, collected 0 and 24 h after each treatment,
were sectioned and incubated for 1 h, at 258C, in darkness,
with 10 mM 4,5-diamino¯uorescein diacetate (DAF-2 DA;
CALBIOCHEM, La Jolla, CA, USA) prepared fresh in
distilled water or in PBS. This probe is highly speci®c for
NO (Kojima et al., 1998) and has been successfully tested in
plant cells (Pedroso et al., 2000a, b). After incubation, the
samples were washed twice in water, mounted in water or
Vectashield, and observed with a ¯uorescence or confocal
microscope. DAF-2 ¯uoresces green (excitation 495 nm;
emission 515 nm; long-pass ®lter of 515 nm; Kojima et al.,
1998). Unstained leaf sections and sections stained after
boiling (dead) were used as controls. For quantitation, at
least six clonal leaves were collected per treatment, and
stained cells were counted within three to ®ve sections per
leaf. Values are the mean of six independent experiments.
Induction and inhibition assays
Chemical agents (sodium nitroprusside and NG-monomethyl-L-arginine) and hypergravity were used to investigate the involvement of NO on DNA fragmentation and
cell death in KalanchoeÈ leaves. Leaves were incubated for
3 h, at 1 g, in ®lter sterilized MS/2 medium with 10 ÿ4 M
sodium nitroprusside (SNP), a NO donor; leaves incubated
in medium without SNP were used as controls. Incubation
was performed in a shaker at 60 rpm, at 23 + 28C.
In independent assays, leaves were exposed to hypergravity (150 g) for 3 h in ®lter sterilized MS/2 medium with
and without (control) 0.5 mM NG-monomethyl-L-arginine,
L-NMMA, a competitive NO-synthase inhibitor. Exposure
985
in medium with 0.5 mM D-NMMA, which has no
signi®cant e€ect on NO-synthase, was performed as
negative control. Leaves kept for 3 h at 1 g under agitation
were used as an additional control. After 3 h, leaves were
washed in sterile fresh medium without SNP or NMMA
and immediately assayed for NO visualization or kept in
culture overnight, for the detection of apoptosis (see
above). Double staining for simultaneous detection of NO
and DNA fragmentation was also tested; live cells and leaf
sections were stained with DAF-2 DA, ®xed, and then
processed for TUNEL assay and DAPI counterstaining.
This staining was possible because nuclei do not stain with
DAF-2 DA; even so controls should be performed for every
step of the process. Assays in the presence of other NO
donors, NO-synthase inhibitors, NO scavengers, nitrite or
nitrate supplementations were not included in the present
study as the results are described elsewhere (Pedroso et al.,
2000a, b).
Fluorescence and confocal microscopy
Quantitation of NO formation, DNA fragmentation and
cell death was performed on a Nikon (Tokyo, Japan)
inverted microscope equipped with UV (excitation 360 nm;
emission 420 nm) and FITC (excitation 450±490 nm;
emission 520 nm) ®lters and a Nikon camera with 400
ASA Kodak Ektachrome ®lm. Confocal images were
obtained using Leitz Fluor 40 and 100 1.3NA oil
PL FLUOTAR objective lenses at a Leica TCS-NT
confocal laser scanning microscope (Leica Lasertechnik
GmbH, Heidelberg, Germany) equipped with Argon/
Kripton and UV lasers (excitation spliter DD488/586;
RSP500), and Leica TCS-NT software (TCS-NT version
1.5.451). Serial confocal optical sections were taken at a
step size of 1 mm. Molecular Dynamics ImageSpace software (Molecular Dynamics, Inc., Sunnyvale, CA, USA)
was used to create 3-D projections of serial confocal optical
sections, merged or separated two- or three-channel 3-D
images, and sequences of 3-D images at di€erent angles to
simulate object rotation. Images were contrast enhanced
using image processing software (Photoshop1; Adobe
Systems, Inc., Mountain View, CA, USA) and printed on
a Kodak ds 8650P Color Printer (Eastman Kodak
Company, Rochester, NY, USA).
R E S ULT S
Detection of DNA fragmentation and cell death in di€erent
gravity environments
The appearance and intensity of the ¯uorescence after
¯uorescein-dUTP labelling of free 30 -OH termini (TUNEL;
green; FITC channel) was indicative of the extent of DNA
fragmentation present in leaf cells following a gravitational
force stimuli. To determine accurately the percentage of cell
death, all cells were counterstained with DAPI after
TUNEL assay and observed in the UV and FITC channels
or, in addition, the TRITC channel. No separate UV
channel images are presented in this work. Cells with
DAPI-positive nuclei (blue; UV channel) showing no green
986
Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis
¯uorescence in the FITC channel (results indicative of the
absence of DNA breaks), were considered as `nonapoptotic'. Cells with simultaneously bright green
TUNEL-positive and DAPI-negative nuclei (in the FITC
and UV channels, respectively), features indicative of
irreversible DNA damage, and presenting cytological
features of PCD, were designated as `apoptotic'
(Fig. 1A±H). These cells were quanti®ed as death cells.
Positive and negative controls of the TUNEL assay further
con®rmed the absence of artifacts; TUNEL was positive for
both nuclei and chloroplast nucleoids in leaf sections
treated with DNase-I ( positive controls); some nuclei were
also DAPI-positive. In negative controls (leaf sections
incubated without TUNEL enzyme), only DAPIpositive nuclei and some DAPI-positive nucleoids were
observed. Figure 1E shows a merged confocal image of a
Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis
non-apoptotic, double stained mesophyll cell. Most chloroplasts, showing starch grains and bright red auto¯uorescence (chlorophyll), were distributed close to the cell wall,
far from the nucleus (Fig. 1E).
Cell death in leaves is di€erentially a€ected by di€erent
gravity environments
Culture in 2 10 ÿ4 g (clinorotation; simulated hypogravity), 1 g (Earth's gravity; controls) and 20 and 150 g
(hypergravity), in darkness or a 16 h photoperiod, showed
that the gravity environment signi®cantly a€ects DNA
fragmentation and cell death in K. daigremontiana leaves,
for both plantlets and isolated leaves. In this report we
emphasize the results obtained with isolated leaves.
Figures 1A±C and 2 show that the percentage of leaf cells
exhibiting irreversible DNA fragmentation (TUNELpositive cells) increased proportionally with the increase
of gravitational force, from 2 10 ÿ4 g to 150 g. Labelled
cells were detected in leaf epidermis (mainly guard cells),
parenchyma and tracheary elements undergoing terminal
di€erentiation (Fig. 1A±D). Guard cells (Fig. 1F) and
subepidermal parenchyma cells (Fig. 1H) were the most
a€ected by gravity changes. Leaf culture in darkness, in
hypogravity or 1 g, increased the number of TUNELpositive cells. However, di€erences between leaf culture in
darkness or light were not signi®cant (P 5 0.05) (Fig. 2).
Except for later stages of cell death, the pattern of
nuclear degeneration was identical for clinorotated, control
and centrifuged cells. Early stages of apoptosis were
characterized by bright green TUNEL-positive nuclei with
the same shape as DAPI-positive ones (Fig. 1D and F).
Disorganization of the nuclear envelope and marginalization of chromatin were also observed in subsequent stages
of the process (Fig. 1H and I), and con®rmed by
transmission electron microscopy (data not shown). At
1 g, bright areas of labelled DNA fragments were
frequently concentrated at the periphery of the nucleus,
987
while dark areas of fully degraded nuclear material
appeared in the nucleus core (Fig. 1I). TUNEL-positive
chloroplasts, close to the nucleus, with one to seven bright
green ¯uorescent spots (indicative of nucleoid DNA
fragmentation) were always observed in apoptotic guard
cells (Fig. 1F), and in parenchyma cells undergoing
apoptosis (Fig. 1H and L).
In contrast to apoptotic cells in 1 g (Fig. 1I), the
apoptotic cells exposed to hypergravity presented drastic
nuclei condensation and blebbing, resulting in nuclei
frequently surrounded by bright green ¯uorescent bodies
(Fig. 1G and J). 3-D projections of serial confocal optical
sections from images identical to those on Fig. 1G and J
showed several stages of bleb formation on the nuclear
surface (image not shown). TUNEL-positive globular
bodies were observed close to the apoptotic nucleus, both
in hypergravity- and hypogravity-exposed leaf cells (Fig. 1J
and K). Figure 1J shows those bodies detaching and
moving away from a condensed nucleus. In epidermal
cells, the migration of those nuclear bodies to the periphery
of the cell, forming `lines' of bright spots, was frequently
observed (Fig. 1G). Transmission electron images con®rmed the nuclear blebbing observed and formation of
vesicles containing nuclear materials (images not shown).
Degeneration of the nuclear envelope and of mitochondria
around the nucleus was also common. In hypergravity, the
appearance of a second nucleolus before nuclear condensation was also observed (image not shown).
In simulated hypogravity, nucleus fragmentation in two
portions was commonly observed for elongated nuclei
(Fig. 1K), with or without the presence of nuclear bodies.
Di€erential TUNEL reaction with chloroplast di€erentiation
and gravitational force
Based on colour, starch content and TUNEL reaction,
chloroplasts were divided into three types: (1) bright red,
with or without starch grains, TUNEL-negative (see
F I G . 1. In situ detection of nuclear and chloroplastidial DNA fragmentation and cell death (apoptosis) in KalanchoeÈ daigremontiana leaves. A±C,
Fluorescence microscope images of leaf sections obtained using a FITC ®lter (excitation 450±490 nm; emission 520 nm) after TUNEL assay to
detect DNA fragmentation (green ¯uorescence). TUNEL-positive nuclei are in green; red is due to chlorophyll auto¯uorescence. Leaves were
collected and processed for TUNEL (see Materials and Methods) after 15 d exposure to di€erent levels of gravity stimulation. The di€erences
observed between treatments were identical for di€erent focal planes. A, Hypogravity, 2 10 ÿ4 g; B, Earth's gravity, 1 g (control); C,
hypergravity, 20 g. Bar ˆ 20 mm. D±O, Confocal laser scanning microscopy images acquired after TUNEL assay (FITC channel) and DAPI
counterstaining (UV channel); red, when present, is due to chlorophyll auto¯uorescence (TRITC channel). D, DAPI- (blue) and TUNEL-positive
nuclei (green) in a cross-section from a leaf exposed to 20 g, for 15 d (merged image; UV-FITC). Bar ˆ 11 mm. E, Non-apoptotic, double stained
leaf parenchyma cell from a leaf cultured in 1 g, showing a DAPI-positive nucleus and TUNEL-negative chloroplasts (red auto¯uorescence only)
(3-D projection; UV-FITC-TRICT). Bar ˆ 5 mm. F, Apoptotic guard cells showing nuclear and chloroplastidial DNA fragmentation (green
spots) at 20 g (merged image, UV-FITC channels only to eliminate chlorophyll auto¯uorescence). Bar ˆ 3.5 mm. G, Nuclear DNA fragmentation
in an epidermal cell. Note the presence of nuclear bodies (bright spots) migrating towards the cell wall at 20 g (merged image, UV-FITC).
Bar ˆ 3.0 mm. H, Nuclear and chloroplastidial DNA fragmentation in mesophyll cells after 15 d of exposure to 150 g (3-D projection; UV-FITCTRITC). Bar ˆ 11 mm. I, Pseudo-colour image of an apoptotic nucleus from a leaf cell cultured in 1 g. Note the marginalization of the chromatin.
Hot colours correspond to areas of high DNA fragmentation (FITC channel; no ¯uorescence on UV and TRICT channels). Bar ˆ 1.2 mm. J,
Example of an apoptotic nucleus from a cell of a leaf cultured in hypergravity. Note the presence of nuclear bodies detaching from the condensed
nucleus (UV-FITC-TRITC). Bar ˆ 1.7 mm. K, Example of an apoptotic nucleus from a cell of a leaf cultured in simulated hypogravity. Note the
nuclear fragmentation and presence of nuclear bodies (UV-FITC-TRITC). Bar ˆ 1.6 mm. L, TUNEL-positive nucleoids (green spots) in
chloroplasts of mesophyll cells after 15 d of leaf exposure to hypergravity (3-D projection; UV-FITC-TRITC). Bar ˆ 2.2 mm. M±O, TUNELpositive cell from a leaf exposed to hypergravity (3-D projection; UV-FITC). Images arti®cally coloured to enhance contrast. M, Note distribution
of chloroplasts around the nucleus and the presence of a network of connections between chloroplasts (arrows). Bar ˆ 6.8 mm. N, Linked
TUNEL-positive chloroplasts concentrate around the nucleus or irradiate toward the cell wall, as shown by the red lines drawn over the image.
Bar ˆ 31 mm. O, This image shows the intricate network of connections (yellow lines) observed between labelled and unlabelled chloroplasts.
Yellow lines were drawn over the 3-D projection series in M. Bar ˆ 26 mm.
Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis
350
80
Immediately after exposure
A
nuclei
nucleoids
NO
300
250
200
60
40
150
20
0
<1 g <1 g
Light Dark
1g
1g
Light Dark
20 g 150 g
Dark Dark
Physical conditions tested
F I G . 2. Cell death (TUNEL-positive cells) in the epidermis and
mesophyll of KalanchoeÈ leaves after 15 d exposure to di€erent levels of
gravity stimulation (2 10 ÿ4, 1, 20 and 150 g) and light conditions
(darkness and a 16 h photoperiod). Leaves were ®xed immediately after
each treatment, sectioned, processed for TUNEL and counterstained
with DAPI. Leaves cultured at 1 g, in darkness and light, were used as
controls. Leaf sections treated with DNase-I after ®xation and
permeabilization ( positive control) displayed 85 % TUNEL-positive
cells. Leaf sections processed without addition of terminal transferase
did not label. DAPI-positive vs. TUNEL-positive cells in the epidermis
(h) and mesophyll (j) were counted in ten individual microscope ®elds
from at least six leaf sections from each of the four leaves randomly
collected per treatment per experiment. Values are the means of four
independent experiments. Error bars are lower than 8 %.
Fig. 1E); (2) orange, with starch grains, TUNEL-positive
(Fig. 1H and L); and (3) bright orange ¯uorescence at the
periphery, almost completely ®lled with starch, TUNELnegative.
The number of labelled chloroplasts increased with
increasing gravitational force. Labelled chloroplasts were
always present when nuclei presented severe DNA fragmentation (Fig. 1H and L). However, at lower gravitational
forces, including 20 g, subepidermal parenchyma cells
presenting only labelled chloroplasts were frequently
observed (image not shown).
3-D projections from sequence series of optical sections
(Fig. 1M±O) showed the presence of straight connections
among chloroplasts of apoptotic cells (Fig. 1M), not
observed in non-apoptotic cells. Labelled chloroplasts
were concentrated around the nucleus and close to the
cell wall. Distribution of chloroplasts around the nucleus
was not observed in non-apoptotic cells. In Fig. 1N, red
lines were drawn over the projection series of optical
sections to show the TUNEL-positive chloroplasts connected with each other, from the cell wall to the nucleus.
3-D imaging analysis further revealed an intricate network
of connections between labelled chloroplasts and TUNELnegative ones (Fig. 1O).
Nuclear DNA fragmentation is preceded by nucleoid DNA
fragmentation
In order to determine how fast leaves responded to
changes of gravity, in situ detection of DNA fragmentation
was performed in clonal leaves immediately (0 h) and 24 h
after 10, 30 and 60 min at 150 g. Ten minutes' (T10)
exposure to 150 g drastically increased the number of
100
50
0
0
10
30
80
70
60
50
40
30
20
10
0
60
Hyper-G exposure (min)
300
B
250
200
24 h after exposure
nuclei
nucleoids
NO
150
100
50
0
0+24 h
10+24 h
30+24 h
80
70
60
50
40
30
20
10
0
60+24 h
Percent of cells with NO
100
TUNEL-positive cells/leaf section
TUNEL-positive cells (%)
988
Hyper-G exposure (min) +24 h at 1 g
F I G . 3. Quantitation of nitric oxide (NO) formation and of nuclear
and chloroplast DNA fragmentation following acute hypergravity
treatments. A, Leaves, collected at the times indicated after hypergravity exposure (150 g; hyper-G), were sectioned, stained for 1 h with
10 mM DAF-2 DA for nitric oxide visualization, ®xed, processed for
TUNEL and counterstained with DAPI. Leaf sections, fresh and ®xed,
not assayed for TUNEL, treated with DNase-I, processed without
terminal transferase, and without DAPI staining, were used as controls
of this staining assay. B, Hyper-G treated leaves, kept for 24 h in 1 g
under a 16 h photoperiod, were processed as described for A. Leaves
not exposed to hypergravity (0 ‡ 24 h) were also collected and used as
controls. Three to ®ve leaf sections (90 mm), performed in at least six
clonal leaves per treatment, were used to quantify the percentage of leaf
cells with NO (shaded line) and, within TUNEL-positive cells, those
presenting nucleoid (m) and nuclear (j) DNA fragmentation. Values
are the mean of six independent experiments. Error bars are lower
than 7 %.
TUNEL-positive cells. This increase was entirely due to
nucleoid DNA fragmentation, as the number of cells with
nuclei presenting DNA fragmentation was identical to 1 g
controls (Fig. 3A). Guard cells and subepidermal parenchyma cells were the most a€ected. Longer exposures, T30
and T60 min, further increased nucleoid DNA fragmentation (Fig. 3A). DNA fragmentation in brightly DAPIstained nuclei also increased, compared to T0 and T10 min,
mainly in epidermal cells (Fig. 3A).
Detection of DNA fragmentation 24 h after leaf
exposure to 150 g showed that the number of TUNELpositive nuclei and the extent of nuclear DNA fragmentation had further increased compared to the previous day
(Fig. 3B). The nuclei were no longer DAPI-positive, the
number of cells with TUNEL-positive nucleoids decreased
signi®cantly (Fig. 3B), and death cells were detected in all
samples. The highest number of labelled chloroplasts and
Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis
989
F I G . 4. Visualization of nitric oxide (NO) and DNA fragmentation in K. daigremontiana leaves exposed to hypergravity. A, Presence of NO in
epidermal cells (green ¯uorescence) of a leaf incubated for 3 h at 150 g, stained with 10 mM DAF-2 DA. In controls, only a few cells were stained
(see Fig. 5). Fluorescence microscopy image (excitation 450±490 nm; emission 520 nm). Bar ˆ 20 mm. B, Detail of guard cells stained for NO.
Note the absence of staining in vacuoles (v) and the yellow ¯uorescence in the chloroplasts due to merging of confocal images from the FITC
(green; NO staining) and TRITC (red; chlorophyll auto¯uorescence) channels. Bar ˆ 3.4 mm. C, Fluorescence microscope image of a mesophyll
section of a centrifuged leaf stained for NO as described in A. Note the presence of NO (green) in some chloroplasts while others show no NO
production (star). Bar ˆ 12.5 mm. D, Magni®ed confocal image (merged FITC-TRITC) of C. DAF-2 DA-positive chloroplasts in green.
Bar ˆ 2 mm. E, Simultaneous detection of NO and DNA fragmentation in a mesophyll cell by DAF-2 DA and TUNEL, respectively. This was
possible as nuclei do not stain with DAF-2 DA. Note that chloroplasts strongly stained for NO are close to the cell wall, while those close to the
nucleus are not stained (circular shadows). Nuclear DNA fragmentation is detected, although the nucleus was still DAPI-positive (image not
shown). 3-D projection, only the FITC channel is shown. Bar ˆ 6.7 mm. F±G, Merged confocal images of mesophyll chloroplasts stained for NO.
Note NO localization (green) around starch grains (arrows). Red is due to chlorophyll auto¯uorescence. F, FITC-TRITC channels.
Bar ˆ 3.6 mm. G, TRITC-transmission. Bar ˆ 3.6 mm. H, Merged confocal image showing the connections between DAF-2 DA-positive and
negative chloroplasts (asterisk). This image suggests that NO di€usion might occur between chloroplasts. Yellow results from the merging of
FITC-TRITC channels. Bar ˆ 2.8 mm.
nuclei were recorded for T60 and T60 ‡ 24 h, respectively
(Fig. 3A and B), in subepidermal parenchyma cells.
Nitric oxide formation precedes and correlates with DNA
fragmentation
DAF-2 DA staining (green) of fresh leaves immediately
and 24 h after 10, 30 and 60 min in hypergravity (150 g),
showed that NO formation signi®cantly increased after
exposure, decreasing to levels higher than those detected for
controls 24 h later (Fig. 3A and B). NO was detected
di€used in the cytosol of epidermal cells, mainly in guard
cells (Fig. 4A and B), or it was just detected in chloroplasts
(Fig. 4C and D). Sectioned and stained controls (leaves in
1 g for the same period of time) con®rmed that the NO
burst detected was a consequence of hypergravity exposure
and not of sample manipulation.
To investigate whether NO formation was involved in
DNA fragmentation, leaves were incubated for 3 h in 1 g,
in medium with and without sodium nitroprusside (SNP), a
NO donor, and in hypergravity, in medium with and
without NG-mono-methyl-L-arginine (L-NMMA), a
NO-synthase inhibitor. Incubation in hypergravity in
medium with D-NMMA, which has no signi®cant e€ect
on NO-synthase, was performed as negative control. Leaves
collected immediately after the treatments were sectioned,
stained with DAF-2 DA, processed for TUNEL, and then
counterstained with DAPI. Figure 5 shows the results
obtained immediately after treatment. SNP and hypergravity both increased the number of cells showing NO and
DNA fragmentation, compared to 1 g controls (Fig. 5).
Culture in hypergravity with L-NMMA signi®cantly
decreased NO formation and DNA fragmentation, in
both epidermal and mesophyll cells, compared to centrifuged samples without the inhibitor, and to 1 g controls
(Fig. 5). NO formation and DNA fragmentation was
identical for samples cultured in hypergravity with and
without D-NMMA (data not shown), recon®rming the
inhibitory e€ect of L-NMMA. These results showed that
NO formation was directly correlated with DNA fragmentation. Additional assays in which leaves were ®xed and
processed for TUNEL without previous in vivo incubation
990
Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis
for 1 h in DAF-2 DA, showed that nucleoid DNA
fragmentation increased during the ®rst hour immediately
after exposure (data not shown). Figure 5 shows that the
inhibition of NO formation by L-NMMA was more
e€ective in chloroplasts (empty bars; mesophyll cells) than
in the cytosol (shaded bars; epidermal cells).
3-D projections showed that chloroplasts brightly stained
for NO were concentrated close to the cell wall (Fig. 4E),
whereas dimly stained chloroplasts converged to the
nucleus. The chloroplasts next to the nucleus were not
stained (Fig. 4E, dark shadows on the nucleus). At that
moment (1 h after the 3 h treatment), DNA fragmentation
was already detected in the nucleus (Fig. 4E), although
DAPI ¯uorescence was still bright in the UV channel
(image not shown).
NO was detected di€used in the stroma of the DAF-2
DA-positive chloroplasts preferentially around starch
grains (Fig. 4F±H). The red auto¯uorescence of the
chlorophyll frequently surrounded, completely or partially,
the green ¯uorescent core (Fig. 4F and G). The appearance
of a yellow colour after merging the confocal images from
the FITC and TRITC channels con®rmed that NO could
also be detected close to the thylakoid membranes
(Fig. 4H).
DAF-2 DA-positive connections between stained-stained
and stained-unstained chloroplasts were also observed
(Fig. 4H). However, in the latter, the side of the tubular
connection in contact with the unstained chloroplast was
not ¯uorescent, suggesting the possibility of NO di€usion
between chloroplasts.
Identical results were obtained for the other hypergravity
treatments. As for the TUNEL assay, it was the epidermal
Percent of TUNEL-positive cells
0
5
0
10
10
15
20
25
30
35
40
45
Control
10-4 SNP
Hyper-G
Hyper+
L-NMMA
20
30
40
50
60
70
80
90 100
Percent of NO stained cells
F I G . 5. Induction or inhibition of nitric oxide (NO) formation and
DNA fragmentation by chemical agents and centrifugation. KalanchoeÈ
leaves were incubated for 3 h in 1 g in culture medium with or without
10 ÿ4 sodium nitroprusside (SNP), a nitric oxide donor, or at 150 g,
with or without NG-mono-methyl-arginine (NMMA), a NO-synthase
inhibitor, to determine the e€ect of NO on DNA fragmentation.
Leaves were sectioned immediately after each treatment, stained for 1 h
with 10 mM DAF-2 DA for nitric oxide visualization, ®xed, processed
for TUNEL to detect DNA fragmentation and counterstained with
DAPI. TUNEL-positive cells (j; epidermis and mesophyll) and DAF2 DA-positive cells in mesophyll (NO di€used in the chloroplasts; h)
and in epidermis (NO di€used in the cytosol; D) were quanti®ed as
described for Fig. 2. Values are the mean of six independent
experiments. Error bars are lower than 8 %.
(mainly guard cells) and subepidermal parenchyma cells
(chloroplasts) that were preferentially stained for NO
(Fig. 4A±D).
All apoptotic cells were positive for NO, but not all NOpositive cells were/became apoptotic. In all assays, leaf-cell
response was identical for both leaves and plantlets. In
both, leaf-plantlet formation paralleled the cell death
increase with the increase of gravitational force (results
not shown).
DISCUSSION
The present results provide microscopic and TUNEL
staining evidence that cell death is induced in response to
gravity changes. Cell death increased with increased levels
of gravity stimulation, and was shown to be increased or
reduced by chemical agents (SNP or L-NMMA, respectively). Plantlet and leaf culture for 15 d under multiples of
Earth's gravity was expected to induce a stress response in
leaf cells, which could eventually lead to cell death. These
results are in accordance with previous reports showing that
centrifugation and other forms of mechanical stress can
lead to cell damage and/or cell death (Pedroso and Durzan,
1998, 1999; Magalhaes et al., 1999). Cell death was
increased by exposure to a NO generator (SNP) and
reduced by a NO-synthase inhibitor (L-NMMA). Addition
of D-NMMA did not a€ect cell death. This indicates that
the cell death process induced was under physiological
control and, possibly, mediated by NO.
In both hyper- and hypogravity, cell death was characterized by distinct chromatin marginalization, nuclear
condensation and blebbing, resulting in the formation of
nuclear-apoptotic bodies. These are typical features of
apoptosis in animal (Bowen et al., 1996) and plant cells
(Havel and Durzan, 1996a; Wang et al., 1996; Yeng and
Yang, 1998). In simulated hypogravity, nuclei often broke
into two fragments (see Fig. 1K), a phenomenon not
observed in cell death induced by hypergravity (see
Fig. 1J). We did not determine the cause of this di€erence.
Nuclear breakage after formation of small nuclear-apoptotic bodies might represent a later stage of nuclear
degeneration. Based on the above, these results provide
strong evidence that the cell death process induced in
KalanchoeÈ leaves was apoptosis.
The in vivo visualization of NO by DAF-2 DA required
an incubation period of 1 h, whereas determinations of
DNA fragmentation were made on destructively sampled
leaves. Nevertheless, using acute hypergravity treatments
(10±60 min) and several experimental controls, it was
possible to show that the NO bursts were caused by
exposure to hypergravity and not by sample manipulation
and/or sectioning, and that they preceded chloroplast DNA
fragmentation. Chloroplasts were the ®rst visible organelles
to show NO and DNA fragmentation. K. daigremontiana
chloroplasts have small, uniformly dispersed nucleoids in
the matrix between the thylakoid membranes and/or the
grana stalks (Kuroiwa et al., 1981). Treatments with SNP
and L-NMMA enabled us to show a direct correlation
between NO and DNA fragmentation in chloroplasts and
in tissues (epidermis and mesophyll). Identical results were
Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis
recently reported for Arabidopsis thaliana (Garces et al.,
1999) and Taxus brevifolia (Pedroso et al., 2000b). No
DAF-2-positive cells or DNA fragmentation were observed
when centrifugation was performed in the presence of
carboxy-PTIO, a NO scavenger (Pedroso and Garces, pers.
comm.). The consistent staining of KalanchoeÈ chloroplasts
with DAF-2 DA suggests that NO was being produced in
these organelles, and that NO was directly or indirectly
responsible for the damage of chloroplast DNA. This
reinforces previous results on the possible involvement of
free radicals in signal pathways leading to PCD (Jacobsen,
1996; Panavas and Rubinstein, 1998), and supports the
involvement of NO in DNA damage and plant cell death,
as described for animal systems (Wink et al., 1991; BruÈne
et al., 1998; Tannenbaum, 1998).
991
Chloroplast DNA fragmentation was detected 10 min
after exposure to hypergravity. Nuclear DNA fragmentation was ®rst detected 20 min later (at 30 min of exposure),
increasing until 24 h after the treatments, when chloroplast
degeneration was severe. In our study, at 30 and 60 min of
exposure, the nuclei that labelled for TUNEL were also
brightly stained for DAPI. This indicates that most of their
DNA was still intact. After 24 h, no intact DNA was
detected on TUNEL-positive nuclei. Morphological
changes occurring in chloroplasts before nuclear changes
were also reported for resistant tobacco plants infected with
TMV (Mittler et al., 1996), and during xylogenesis in
Zinnia cells (Fukuda et al., 1998). Our results are also in
accordance with those described for PCD in tracheary
elements, a process estimated to be completed in 8 to 12 h
(Groover and Jones, 1999), and for toxin-induced cell death
in asc/asc tomato protoplasts (6 to 16 h) (Wang et al.,
1996).
In mesophyll cells, environmental stresses are readily
perceived by chloroplasts and elicit an oxidative response
(NoodeÂn, 1988; Allen, 1993; BruÈggemann et al., 1994;
Green and Fluhr, 1995; Allen and Raven, 1996; Allan and
Fluhr, 1997; Buchanan-Wollaston, 1997). NO reacts with
superoxide (O2ÿ ) to form peroxynitrite (OONO ÿ ) (Pryor
and Squadrito, 1995), which damages DNA, RNA,
proteins and lipids (Stamler et al., 1992). Thus it is not
surprising that chloroplast-containing cells closest to the
region of stimuli/stress application (guard cells and
subepidermal parenchyma cells) were the most sensitive to
external changes (light or gravity). The increase in cell death
recorded in darkness was also probably related to
senescence in photosynthetic tissues (Panavas and
F I G . 6. Summary of the time sequence of events leading to cell death in
KalanchoeÈ mesophyll cells as a response to a change in gravity. A, Leaf
exposure to higher levels of gravity stimulation (hypergravity) induces a
NO burst (green) in the chloroplasts (Ch; red) of mesophyll cells that
are localized close to the cell wall (Cw), on the site of force application.
The NO burst precedes and correlates with nucleoid DNA fragmentation during the following 3 h. No nuclear DNA fragmentation is
detected (blue; DAPI). A bright red auto¯uorescence from chlorophyll
is present. B, After 4 h, the number of chloroplasts stained for NO is
still high, nucleoid DNA fragmentation is highest and nuclear DNA
fragmentation is detected in DAPI-positive nuclei (DAPI-TUNEL).
Chloroplasts redistribute. Unstained chloroplasts move toward the
nucleus. In the following hours, the auto¯uorescence of TUNELpositive chloroplasts close to the cell wall becomes orange, indicative of
a reduction of chlorophyll content, and a network of connections
between chloroplasts becomes apparent. C, After 9±12 h, the number
of chloroplasts stained for NO is reduced and microscopic evidence of
severe chloroplast degeneration is present. TUNEL-positive chloroplasts, when present, are localized close to apoptotic nuclei. An
intricate network of connections between chloroplasts is observed.
Nuclear condensation and blebbing with formation of apoptotic bodies
is detected. The time required for completion of B and C varies with
the intensity and duration of the stimulus. Cytological evidence
indicates that the process of cell death by apoptosis can be completed
in 9 h. S and r represent `turning points' in the process, where S are
cells that survive and adapt to the stimulus applied and proliferate,
while r represents cells that through addition of L-NMMA could be
rescued from phase A and prevented from dying. This suggests the
existence of at least a checkpoint (*) between A and B, and indicates
that the process can be manipulated within the ®rst hours following the
application of the stressful stimulus.
992
Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis
Rubinstein, 1998). A direct consequence of the exposure to
increasing levels of gravity stimulation was the increase and
spread of the population of TUNEL-positive parenchyma
cells to deeper cell layers.
Because nitrate was used in the culture medium, we
cannot exclude the conversion of nitrite to NOx
(NO ‡ NO2) by nitrate reductases, and/or non-enzymatic
ones, resulting from chemical reactions between plant
metabolites and accumulated NO2ÿ and/or decomposition
of nitrous acid (Dean and Harper, 1986, 1988; Klepper,
1990; Vliet et al., 1997; Durner et al., 1998; Yamasaki et al.,
1999). The preferential localization of DAF-2 DA staining
around the starch grains (see Fig. 4F) might indicate nonenzymatic NO generation (Nishimura et al., 1986). However, the inhibitory e€ect of L-NMMA on NO production
(see Fig. 5) led us to reject this possibility. Our results
suggest the presence of an inducible NO-synthase in
KalanchoeÈ leaf cells. The recent report of a gene in
Arabidopsis thaliana with sequence similarity with animal
NO-synthase (Mayer et al., 1999), further supports this
assumption. The localization of the NO-staining, in the
cytosol and/or chloroplasts coincides with sites of known
location of soluble L-arginine (Ludwig, 1993), and those
previously reported in in situ localization assays (Barroso
et al., 1999; Pedroso et al., 2000a, b).
The detection of NO and DNA breaks in chloroplasts
was accompanied by a decrease in chlorophyll auto¯uorescence, increase in starch content, and followed by severe
nuclear DNA damage. Chloroplast degeneration was
con®rmed by electron microscopy and by a concomitant
loss of photosynthetic capacity (unpubl. res.). Membrane
lipids of chloroplasts could be one of the primary targets
for NO attack, detrimentally a€ecting the eciency of
photosynthesis (Leshem et al., 1997). The di€erential
TUNEL reaction with chloroplast di€erentiation and
gravitational forces was probably due to the `redox state'
of the plastid at the moment of stimuli/stress exposure and
its ability to cope with an additional stress (Allen and
Raven, 1996).
TUNEL-positive chloroplasts were linked by cytoplasmic
connections and concentrated around the nucleus (see
Fig. 1M). Linkages irradiated in lines to the cell wall (see
Fig. 1N). Exchange of protein molecules through connections between chloroplasts was recently demonstrated
(KoÈhler et al., 1997; Shiina et al., 2000). Confocal images
from parenchyma cells double stained with DAF-2 DATUNEL (see Fig. 4E), suggested that NO di€used along a
cytoplasmic network to the nucleus where endonuclease
activity was then initiated.
How the chloroplast stress responses leading to cell death
were related to the network of connections remains unclear.
This intricate network of connections was similar to a
cortical endoplasmic reticulum (see Fig. 1M) (Gunning and
Steer, 1996). The network could engulf NO-producing
chloroplasts to prevent further cellular DNA damage, and
promote calcium in¯ux favouring DNA damage and
chloroplast elimination. It could also increase molecule
transport among degenerating organelles and the neighbouring cells, prior to cell death. Information transfer to
the nucleus, by direct chloroplast-nucleus or chloroplast-
nucleolus interaction, or by the release of signalling
molecules like octadecanoid 12-oxo-phytodienic acid
(Stelmach et al., 1998), could trigger silent gene-activation
leading to cell adaptation and survival ( for reviews see
Forsburg and Guarente, 1989; Oelmuller, 1989; Taylor,
1989; Zachleder et al., 1989, 1995; Ehara et al., 1990; Susek
and Chory, 1992; Susek et al., 1993; Gillham, 1994).
Cellular degradation products from apoptosis (Havel and
Durzan, 1996a, b) would be salvaged to sustain adaptation,
viability, and ontogeny of the neighbouring cells, assuring
the survival of the species in a harsh(er) environment.
Speci®c checkpoints that regulate plant cell cycle progression in response to oxidative stress (Reichheld et al.,
1999), could explain why some cells undergo programmed
cell death, while others undergo division leading to leafplantlet formation. NO could also play a role in such a
situation depending on its concentration and reactivity with
superoxide concentration (Shen et al., 1998).
At the organ level (leaf), the increases/decreases in NO
formation and cell death were paralleled by increases/
decreases in leaf-plantlet formation (data not shown;
Pedroso and Durzan, 1998). Thus, the involvement of NO
in DNA damage in KalanchoeÈ cells does not exclude a
possible role of NO as a regulator of leaf senescence, as
suggested by Leshem et al. (1998). Senescence leads to a
decrease in chlorophyll content, chloroplast degeneration
(including nucleoid DNA fragmentation) and, ultimately,
nuclear condensation and internucleosomal DNA fragmentation ( for a review see Hensel et al., 1993; Smart, 1994;
Humbeck et al., 1996; Bleecker and Patterson, 1997;
Buchanan-Wollaston, 1997; OrzaÂez and Granell, 1997;
Gray and Johal, 1998), exhibiting cytological features of
PCD by apoptosis (Yeng and Yang, 1998). Our results
suggest that KalanchoeÈ leaves responded to a gravity change
by forming NO, which triggered a senescence-like process
that exhibited characteristics of apoptosis. Simulated
gravity from 2 10 ÿ4 to 150 g, increased NO-related cell
death (Fig. 6). In vitro cultured leaves of K. daigremontiana
provide a unique experimental system for studying the e€ect
of environmental stresses (gravity or other) (Resende and
Pereira-da-Silva, 1965; Pedroso and Durzan, 1999), on a
potential homeostatic balance between plantlet formation
and apoptosis in this species.
In conclusion, cell death in KalanchoeÈ leaves increases
proportionally with the increase of gravitational force, from
2 10 ÿ4 to 150 g. Stressful gravity environments lead to a
NO burst in chloroplasts. In some cells this triggers a
sequence of events that leads to chloroplast DNA damage,
chloroplast degeneration, followed by nuclear degeneration
and cell death by apoptosis.
AC K N OW L E D G E M E N T S
This work was supported by FundacËaÄo para a CieÃncia e a
Tecnologia (contract PRAXIS XXI 3/3.1/CTAE/1930/95),
INVOTAN (3/B/96/PO) and Centro de Biotecnologia
Vegetal (Lisboa, Portugal).
Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis
L I T E R AT U R E C I T E D
Allan AC, Fluhr R. 1997. Two distinct sources of elicited reactive
oxygen species in tobacco epidermal cells. The Plant Cell 9:
1559±1572.
Allen JF. 1993. Control of gene expression by redox potential and the
requirement for chloroplast and mitochondrial genomes. Journal
of Theoretical Biology 165: 609±631.
Allen JF, Raven JA. 1996. Free-radical-induced mutations vs. redox
regulation: cost and bene®ts of genes in organelles. Journal of
Molecular Evolution 42: 482±492.
Barlow PW. 1982. Cell deathÐAn integral part of plant development.
In: Jackson MB, Grout B, Mackenzie IA, eds. Growth regulators in
plant senescence. Wantage, UK: British Plant Growth Regulator
Group, 27±45.
Barroso JB, Corpas FJ, Carreras A, Sandalio LM, Valderrama R,
Palma JM, Lupianez JA, del Rio LA. 1999. Localization of nitricoxide synthase in plant peroxisomes. Journal of Biological
Chemistry 274: 36729±36733.
Bleecker AB, Patterson SE. 1997. Last exit: senescence, abscission and
meristem arrest in Arabidopsis. The Plant Cell 9: 1169±1179.
Bowen ID, Mullarkey K, Morgan SM. 1996. Programmed cell death
during metamorphosis in the blow-¯y Calliphora vomitoria.
Microscopy and Research Technology 34: 202±217.
BruÈggemann W, Klaucke S, Maaskantel K. 1994. Long-term chilling of
young tomato plants under low light. 5. Kinetic and molecular
properties of 2 key enzymes of the Calvin cycle in Lycopersicum
esculentun Mill and L. peruvianum Mill. Planta 194: 160±168.
BruÈne B, Knethen AV, Sandau K. 1998. Nitric oxide and its role in
apoptosis. Nitric Oxide: Extensive Abstracts 1: 1±9.
Buchanan-Wollaston V. 1997. The molecular biology of leaf senescence.
Journal of Experimental Botany 48: 181±199.
Dean JV, Harper JE. 1986. Nitric oxide and nitrous oxide production
by soybean and winged bean during the in vivo nitrate reductase
assay. Plant Physiology 82: 718±723.
Dean JV, Harper JE. 1988. The conversion of nitrite to nitrogen
oxide(s) by the constitutive NAD(P)H-nitrate reductase enzyme
from soybean. Plant Physiology 88: 389±395.
Delledonne M, Xia Y, Dixon RA, Lamb C. 1998. Nitric oxide functions
as a signal in plant disease resistance. Nature 394: 585±588.
Durner J, Wendehenne D, Klessig F. 1998. Defense gene induction in
tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose.
Proceedings of the National Academy of Sciences USA 95:
10328±10333.
Ehara T, Osafune T, Hase E. 1990. Interactions between the nucleus
and cytoplasmatic organelles during the cell cycle of Euglena
gracilis in synchronized cultures. IV. An aggregate form of
chloroplasts in association with the nucleus appearing prior to
division. Experimental Cell Research 190: 104±112.
Ellis RE, Yuan Y, Horvitz HB. 1991. Mechanisms and function of cell
death. Annual Review of Cell Biology 7: 663±698.
Forsburg SL, Guarente L. 1989. Communicationm between mitochondria and the nucleus in regulation of cytochrome genes in the yeast
Saccharomyces cervisiae. Annual Review of Cell Biology 5:
153±180.
Fukuda H, Watanabe Y, Kuriyama H, Aoyagi S, Sugiyama M,
Yamamoto R, Demura T, Minami A. 1998. Programming of cell
death during xylogenesis. Journal of Plant Research 111: 253±256.
Garces H, Pedroso MC, Durzan D. 1999. E€ect of hypergravity on
nitric oxide production and DNA fragmentation in Arabidopsis
thaliana [abstract]. Gravitational and Space Biology Bulletin 13: 19.
Gavrieli Y, Sherman Y, Ben-Sasson SA. 1992. Identi®cation of
programmed cell death in situ via speci®c labeling of nuclear
DNA fragmentation. Journal of Cell Biology 119: 493±501.
Gilchrist DG. 1998. Programmed cell death in plant disease: the
purpose and promise of cellular suicide. Annual Review of
Phytopathology 36: 393±414.
Gillham NW. 1994. Organelle genes and genomes. New York: Oxford
University Press, Inc., 110±365.
Gray J, Johal GS. 1998. Programmed cell death in plants. In:
Anderson M, Roberts JA, eds. ArabidopsisÐAnnual plant reviews
Volume 1. Boca Raton, FL, USA: CCR Press LLC, 360±394.
993
Green R, Fluhr R. 1995. UV-B-induced PR-1 accumulation is mediated
by active oxygen species. The Plant Cell 7: 203±212.
Groover A, Jones A. 1999. Tracheary element di€erentiation uses a
novel mechanism coordinating programmed cell death and
secondary cell wall synthesis. Plant Physiology 119: 375±384.
Gunning BES, Steer MW. 1996. Plant cell biology: structure and
function. London, UK: Jones and Bartlett Publishers, Inc.
Haramaty E, Leshem YY. 1997. Ethylene regulation by nitric oxide
(NO) free radical: A possible mode of action of endogenous NO.
In: Kanellis AK, Chang C, Kende H, eds. Biology and
biotechnology of the plant hormone ethylene. The Netherlands:
Kluwer Academic Publishers, 253±258.
Havel L, Durzan DJ. 1996a. Apoptosis during diploid parthenogenesis
and early somatic embryogenesis of Norway Spruce. International
Journal of Plant Sciences 157: 8±16.
Havel L, Durzan DJ. 1996b. Apoptosis in plants. Botanica Acta 109:
1±10.
Hensel L, Grbic V, Baumgarten DA, Bleecker AB. 1993. Developmental
and age-related processes that in¯uence the longevity and
senescence of photosynthetic tissues in Arabidopsis. The Plant
Cell 5: 553±564.
Humbeck K, Quast S, Krupinska K. 1996. Functional and molecular
changes in the photosynthetic apparatus during senescence of ¯ag
leaves from ®eld-grown barley plants. Plant, Cell and Environment
19: 337±344.
Jacobsen MD. 1996. Reactive oxygen species and programmed cell
death. Trends in Biochemical Science 21: 83±86.
Jacobsen MD, Weil M, Ra€ MC. 1997. Programmed cell death in
animal development. Cell 88: 347±354.
Kerr JFR, Wyllie AH, Currie AR. 1972. Apoptosis: A basic biological
phenomenon with wide-ranging implications in tissue kinetics.
The British Journal of Cancer 26: 239±257.
Klepper L. 1990. Comparison between NOx evolution mechanisms of
wild-type and nr1 mutant soybean leaves. Plant Physiology 93:
26±32.
KoÈhler RH, Cao J, Zipfel WR, Webb WW, Hanson MR. 1997.
Exchange of protein molecules through connections between
higher plants plastids. Science 276: 2039±2042.
Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H,
Hirata Y, Nagano T. 1998. Detection and imaging of nitric oxide
with novel ¯uorescent indicators: diamino¯uoresceins. Analytical
Chemistry 70: 2446±2453.
Kuroiwa T, Suzuki T, Ogawa K, Kawano S. 1981. The chloroplast
nucleus: distribution, number, size, and shape, and a model for
multiplication of the chloroplast genome during chloroplast
development. Plant and Cell Physiology 22: 281±396.
Lancaster JR Jr. 1992. Nitric oxide in cells. American Scientist 80:
248±259.
Leshem YY. 1996. Nitric oxide in biological systems. Plant Growth
Regulators 18: 155±159.
Leshem YY, Haramaty E. 1996. The characterization and contrasting
e€ects of the nitric oxide free radical in vegetative stress and
senescence of Pisum sativum Linn. foliage. Journal of Plant
Physiology 148: 258±263.
Leshem YY, Wills RBH, Ku V-VV. 1998. Evidence for function of the
free radical gasÐnitric oxide (NO.)Ðas an endogenous maturation and senescence regulating factor in higher plants. Plant
Physiology and Biochemistry 36: 825±833.
Leshem YY, Haramaty E, Iluz D, Malik Z, Sofer Y, Roitman L, Leshem
Y. 1997. E€ect of stress nitric oxide (NO): interaction between
chlorophyll ¯uorescence, galactolipid ¯uidity and lipoxygenase
activity. Plant Physiology and Biochemistry 35: 573±579.
Ludwig RA. 1993. Arabidopsis chloroplasts dissimilate L-arginine and
L-citrulline for use as N source. Plant Physiology 101: 429±434.
Magalhaes JR, Pedroso MC, Durzan DJ. 1999. Nitric oxide, apoptosis
and plant stresses. Physiology and Plant Molecular Biology 5:
115±125.
Mayer K, SchuÈller C, Wambutt R et al. 1999. Sequence and analysis of
chromosome 4 of the plant Arabidopsis thaliana. Nature 402:
769±777.
Mittler R, Shulaev V, Seskar M, Lam E. 1996. Inhibition of programmed cell death in tobacco plants during a pathogen-induced
994
Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis
hypersensitive response at low oxygen pressure. The Plant Cell 8:
1991±2001.
Murashige T, Skoog F. 1962. A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiologia Plantarum 15:
473±497.
Nishimura H, Hayamizu T, Yanagisawa Y. 1986. Reduction of NO to
NO2 by rush and other plants. Environmental Science and
Technology 20: 413±416.
NoodeÂn LD. 1988. The phenomenon of senescence and aging. In:
NoodeÂn LD, Leopold AC, eds. Senescence and aging in plants. San
Diego, CA: Academic Press, 1±50.
Oelmuller R. 1989. Photooxidative destruction of chloroplasts and its
consequences for expression of nuclear genes. Planta 167:
106±114.
OrzaÂez D, Granell A. 1997. DNA fragmentation is regulated by
ethylene during carpel senescence in Pisum sativum. Plant Journal
11: 137±144.
Panavas T, Rubinstein B. 1998. Oxidative events during programmed
cell death of daylily (Hemerocallis hybrid) petals. Plant Science
133: 125±138.
Pedroso MC. 1998. Protocols for in vitro culture of Crassulaceae
species in di€erent gravity environments. Report I 3/B/96/PO.
Lisboa: INVOTAN, Instituto de CooperacËaÄo CientõÂ ®ca e TecnoloÂgica Internacional, 15±71.
Pedroso MC. 1999. Detection of nitric oxide and apoptosis in di€erent
gravity environments. Report II 3/B/96/PO. Lisboa: INVOTAN,
Instituto de CooperacËaÄo CientõÂ ®ca e TecnoloÂgica Internacional.
Pedroso MC, Durzan D. 1998. E€ect of hypergravity and simulated
hypogravity on leaf-plantlet development and asexual reproduction of KalanchoeÈ daigremontiana [abstract]. Gravitational and
Space Biology Bulletin 12: 74.
Pedroso MC, Durzan D. 1999. E€ect of hypergravity and simulated
hypogravity on leaf-plantlet formation and asexual reproduction
of KalanchoeÈ daigremontiana. 7th European Symposium on Life
Sciences Research in Space, Maastricht, the Netherlands,
29 May±2 June (in press).
Pedroso MC, Magalhaes JR, Durzan D. 2000a. A nitric oxide burst
precedes apoptosis in angiosperm and gymnosperm callus cells
and foliar tissues. Journal of Experimental Botany 51: 1027±1036.
Pedroso MC, Magalhaes JR, Durzan D. 2000b. Nitric oxide induces
cell death in Taxus cells. Plant Science 157 (in press).
Pennel RI, Lamb C. 1997. Programmed cell death in plants. The Plant
Cell 9: 1157±1168.
Pryor WA, Squadrito GL. 1995. The chemistry of peroxynitrite: a
product from the reaction of nitric oxide with superoxide?.
American Journal of Physiology 268: L699±L700.
Reichheld J-P, Vernoux T, Lardon F, Van Montagu M, Inze D. 1999.
Speci®c checkpoints regulate plant cell cycle progression in
response to oxidative stress. Plant Journal 17: 647±656.
Resende F. 1951. AccËaÄo morfogeÂnica do frio e do fotoperiodismo no
`habitus' vegetativo de Bryophyllum daigremontianum. Boletim da
Sociedade Portuguesa de CieÃncias Naturais (2 SeÂrie) 3: 256±264.
Resende F, Pereira-da-Silva MJ. 1965. Photo- and thermoperiodism.
Their interaction for the phenogenesis. II. About ¯oral impulse in
two species of long-short day (LSDP). Revista de Biologia 5:
203±212.
Ryerson DE, Heath MC. 1996. Cleavage of nuclear DNA into
oligonucleosomal fragments during cell death induced by fungal
infection or by abiotic treatment. The Plant Cell 8: 393±402.
Schwartzman RA, Cidlowski JA. 1993. Apoptosis: the biochemistry
and molecular biology of programmed cell death. Endocrine
Review 14: 133±151.
Shen YY, Wang XL, Wilcken DE. 1998. Nitric oxide induces and
inhibits programmed cell death through di€erent pathways. FEBS
Letters 433: 1125±1131.
Shiina T, Hayashi K, Ishii N, Morikawa K, Toyoshima Y. 2000.
Chloroplasts tubules visualized in transplastomic plants expressing green ¯uorescent protein. Plant and Cell Physiology 41:
367±371.
Smart CA. 1994. Gene expression during leaf senescence. New
Phytologist 126: 419±448.
Stamler JS, Singel DJ, Loscalzo J. 1992. Biochemistry of nitric oxide
and its redox-activated forms. Science 258: 1898±1902.
Stelmach BA, Muller A, Hennig P, Laudert D, Andert L, Weiler EW.
1998. Quantitation of the octadecanoid 12-oxo-phytodienoic acid,
a signalling compound in plant mechanotransduction. Phytochemistry 47: 539±546.
Susek RE, Chory J. 1992. A tale of two genomes: role of a chloroplast
signal in coordinating nuclear and plastid genome expression.
Australian Journal of Plant Physiology 19: 387±399.
Susek RE, Ausubel FM, Chory J. 1993. Signal transduction mutants of
Arabidopsis uncouple nuclear CAB and RBCS gene expression
from chloroplast development. Cell 74: 787±799.
Tannenbaum SR. 1998. E€ects of NO on DNA damage and
DNA synthesis. In: Green IC, ed. Nitric oxide: basic research
and clinical applications. Extensive abstracts. http//www.pasteur.fr/Conf/nitric-abstracts.html.
Taylor WC. 1989. Regulatory interactions between nuclear and plastid
genomes. Annual Review of Plant Physiology and Plant Molecular
Biology 40: 211±233.
Vliet A, Eiserich JP, Halliwell B, Cross CE. 1997. Formation of reactive
nitrogen species during peroxidase-catalyzed oxidation of nitrite.
The Journal of Biological Chemistry 272: 7617±7625.
Wang H, Li J, Bostock RM, Gilchrist DG. 1996. Apoptosis: A
functional paradigm for programmed cell death induced by a hostselective phytotoxin and invoked during development. The Plant
Cell 8: 375±391.
Warden J. 1970a. Leaf-embryo dormency in Bryophyllum crenatum
under short day conditions. Portugaliae Acta BioloÂgica Serie A
Vol. XI, 3±4: 319±338.
Warden J. 1970b. FenoÂmeno de lateÃncia dos embrioÄes foliares de
Bryophyllum crenatum. Alguns aspectos citoquõÂ micos e actividade
morfogeÂnetica. Portugaliae Acta BioloÂgica Serie A Vol XI, 3±4:
385±394.
Wink DA, Kasprzak SK, Maragos CM, Elespuru RK, Misra M,
Dunams TM, Cebula TA, Koch WH, Andrews AW, Allen JS,
Keefer LK. 1991. DNA deaminating ability and genotoxicity of
nitric oxide and its progenitors. Science 254: 1001±1003.
Yamasaki H, Sakihama Y, Takahashi S. 1999. An alternative pathway
for nitric oxide production in plants: new features of an old
enzyme. Trends in Plant Science 4: 128±129.
Yeng C-H, Yang C-H. 1998. Evidence for programmed cell death
during leaf senescence in plants. Plant and Cell Physiology 39:
922±927.
Zachleder V, Kawano S, Kuroiwa T. 1995. The course of chloroplast
DNA replication and its relationship to other reproductive
processes in the chloroplast and nucleocytoplasmic compartment
during the cell cycle of the alga Scenedesmus quadricauda.
Protoplasma 188: 245±251.
Zachleder V, Kuptsova ES, Los DA, Cepak V, Kubin S, Shapizugov JM,
Semenenke VE. 1989. Division of chloroplast nucleoids and
replication of chloroplast DNA during the cell cycle of Dunaliella
salina grown under blue and red light. Protoplasma 150: 160±167.