Effect of Different Gravity Environments on DNA

Annals of Botany 86: 983±994, 2000doi: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 inKalanchoeÈ Leaves

M. C. PEDROSO*{ and D. J. DURZAN{

{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

(Gavrieli et

0305-7364/0

* For corrversity of Ca1819, e-mail m

Di�erent gravity environments have been shown to signi®cantly a�ect leaf-plantlet formation and asexualreproduction in KalanchoeÈ daigremontiana Ham. and Perr. In the present work, we investigated the e�ect of gravityat tissue and cell levels. Leaves and leaf-plantlets were cultured for di�erent periods of time (min to 15 d) in di�erentlevels of gravity stimulation: simulated hypogravity (1 rpm clinostats; 2 � 10ÿ4 g), 1 g (control) and hypergravity(centrifugation; 20 and 150 g). Both simulated hypogravity and hypergravity a�ected cell death (apoptosis) in thisspecies, 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 toacute hypergravity (up to 60 min) showed that chloroplast DNA fragmentation occurred prior to nuclear DNAfragmentation, marginalization of chromatin, nuclear condensation, and nuclear blebbing. Addition of sodiumnitroprusside (NO donor) mimicked centrifugation. NO and DNA fragmentation decreased with NG-monomethyl-L-arginine (NO-synthase inhibitor). The variations in NO levels, nucleoid DNA fragmentation, and cell death show howchloroplasts, cells and leaves may respond (and adapt) to gravity changes. # 2000 Annals of Botany Company

Key words: Apoptosis, chloroplasts, gravitational biology, KalanchoeÈ daigremontiana, mechanical stress, Mother of
Thousands, nitric oxide, programmed cell death, TUNEL.
INTRODUCTION

Traumatic (`accidental') cell death, often called necrosis, isa non-physiological process caused by overwhelming levelsof stresses (e.g. toxins, heat or cold), which involvesdisruption of membrane integrity and subsequent cellularswelling and lysis ( for a review see Schwartzman andCidlowski, 1993; Gray and Johal, 1998). Programmed celldeath (PCD) is a physiological cell death process, dynamic-ally regulated by both developmental and environmentalcues, 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 chroma-tin in the nucleus, nuclear condensation, shrinkage of thecytoplasm, cleavage of nuclear DNA into oligonucleosomes(Ryerson and Heath, 1996), and the breakage of thecytoplasm or nucleus into small, sealed packets, the processis termed apoptosis, both in animals (Kerr et al., 1972) andplants (Havel and Durzan, 1996a; Wang et al., 1996; Yengand Yang, 1998). The in situ detection of DNA fragmenta-tion leading to cell death can be achieved by the terminaldeoxynucleotidyl transferase-mediated dUTP nick endlabelling of DNA 30-OH groups (TUNEL method)

al., 1992; Wang et al., 1996).

0/110983+12 $35.00/00

espondence at: Dept Environmental Horticulture, Uni-lifornia, Davis, CA 95616-8587, USA. Fax �530 [email protected]

In plants, the continuous generation of organ systems bythe plant's meristems is countered by the programmedsenescence and/or abscission (shedding) of existing organsthroughout the life of the plant (Bleecker and Patterson,1997). Senescence involves cell death on a large scale,nutrient recycling and is often interrelated with reproduc-tion ( for reviews see Resende, 1951; Barlow, 1982; Bleeckerand Patterson, 1997; Pennel and Lamb, 1997; Gray andJohal, 1998; NoodeÂ n, 1988). Recent reports have shownthat the process of senescence exhibits features of PCD byapoptosis (in Gray and Johal, 1998; Yeng and Yang, 1998).

KalanchoeÈ daigremontiana Ham and Perr. (Crassulaceae),commonly known as Mother of Thousands, reproducesasexually by forming plantlets in leaf indentations (Warden,1970a, b). In nature, its entire plant body except leaf-plantlets senesces as a consequence of ¯oral di�erentiation(Resende, 1951). The leaf-plantlets fall to earth and convertinto 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 gravitysigni®cantly a�ected leaf-plantlet formation, development,and asexual reproduction in this species (Pedroso andDurzan, 1998). The cytological, morphological and phys-iological changes observed suggested that stressful gravityenvironments (hypergravity) interfered with cellular pro-cesses 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

and under a 16 h photoperiod, were used as controls.

2limiting factors (Pedroso, 1998, 1999).

o

1998, 1999) suggested that, under stressful conditions, leafsenescence could be induced and used as a mechanism forplant survival. As a ®rst step to verify this hypothesis, weinvestigated 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 animportant role as an intra- and intercellular messenger inanimal systems ( for reviews see Lancaster, 1992; Kojimaet al., 1998). In plants, NO formation is associated withethylene biosynthesis (Leshem and Haramaty, 1996),disease resistance (Delledonne et al., 1998; Durner et al.,1998), drought stress (Leshem, 1996; Haramaty andLeshem, 1997), cell death and cell proliferation (Magalhaeset al., 1999; Pedroso et al., 1999, 2000a, b). Evidence for itsfunction as an endogenous maturation and senescenceregulating factor was recently provided (Leshem et al.,1998). Histochemical detection of NO in vivo can now beachieved 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 NOas trigger of cell death in KalanchoeÈ leaves was alsoinvestigated in the present work.

Drastic hypergravity treatments, for short (15 d) andacute (10±60 min) periods of time, were chosen to ascertainthe induction of large-scale cell death ( for a review see Grayand Johal, 1998), helping the cytological distinctionbetween physiological (PCD) and non-physiological (`acci-dental') cell death. Double staining TUNEL-DAPI, micro-scopy techniques, a NO probe (DAF-2 DA), a NO donor(sodium nitroprusside, SNP) and a competitive substrateinhibitor (NG-monomethyl-L-arginine, L-NMMA) of theNO-forming enzyme NO-synthase (EC 1.14.13.39; NO-synthase), were used to show that leaf and leaf-plantletexposure to di�erent gravity environments a�ects leaf PCDin this species, and that variations in the number of cellsshowing DNA damage in chloroplasts and nuclei is directly

984 Pedroso and DurzanÐChlor

correlated with NO formation.

MATERIALS AND METHODS

Plant material and in vitro culture conditions

KalanchoeÈ daigremontiana Ham. and Perr. shoot cultureswere used as a source of leaves and leaf-plantlets (Pedroso,1998). Stock shoot cultures were maintained by micro-propagation on half-strength Murashige and Skoog basalmedium (1962), with 5 mg lÿ1 dithiothreitol, 25 g lÿ1D-glucose, without glycine and growth regulators (MS/2medium); pH was adjusted to 5.5 prior to agar addition(7.5 g lÿ1) and medium sterilization (10ÿ5 Pa, 1208C, for20 min). Subculture was performed every 4 weeks. Unlessotherwise stated, cultures were maintained in glass tubes(150 � 22 mm), at 258C, under a 16 h photoperiodprovided by Phillips F40 AGRO AGROLITE ¯uorescentlamps [maximum radiation at 350±450 nm (blue) and650 nm (red)]. Leaves and leaf-plantlets were cultured onsolid MS/2 medium, on plastic Petri dishes (90 mm) sealedwith transparent ®lm. Leaf-plantlets were cultured in avertical position with their roots in the medium. Leaves
were inoculated horizontally with the upper leaf-page up.
Cultures under hypergravity were kept in darkness, whereasthose exposed to simulated hypogravity were kept indarkness or under a 16 h photoperiod. Leaf-plantlets andleaves cultured under the Earth's gravity (1 g), in darkness

plasts, Nuclei and Apoptosis

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 simulatehypergravity. The centrifuges were modi®ed to preventtemperature oscillations and vibration. Leaves and leaf-plantlets were exposed to 20 and 150 g, for 15 d, or to 150 gfor 10, 30 and 60 min. Clinostats equipped with 1 rpmmotors and adapted to hold a Modular Incubator Chamber(Billups-Rotenberg, Inc., Burlingame, CA, USA) were usedto simulate hypogravity (2 � 10ÿ4 g). Leaves and leaf-plantlets were clinorotated, in darkness and under a 16 hphotoperiod, for 15 d. Immediately after each treatment,samples (leaves) were collected and immersed for 24 h, at48C, in 3.7 % formaldehyde ®xative. Ninety leaf-plantletsand 30 leaves were cultured in each experiment. Exper-iments were repeated four times. In both hypergravity andhypogravity experiments, no ethylene production wasdetected, nor were oxygen, CO , temperature and vibration

In situ detection of DNA fragmentation

Nucleoid and nuclei DNA fragmentation in leaf cells wasdetected in situ using the terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labelling(TUNEL) method (Pedroso, 1998, 1999). Brie¯y, ®xedleaves were hand-sectioned with a razor blade and thesections washed in PBS (10 mM NaH2PO4 , 150 mM sodiumchloride, pH 7.2), ®ve times over 1 h, at room temperature(RT). The sections were then sequentially immersed in anenzymatic solution [4% (v/v) pectinase (Sigma ChemicalCO, St Louis, MO, USA) and 2% (w/v) Cellulase (FlukaBiochemika, CH) in PBS (30 min, at RT); PBS (5 min atRT); methanol in 1% acetic acid (20 min); PBS (5 min atRT), and incubated with 10 mg mlÿ1 proteinase K (Boeh-ringer Mannheim, Germany) in PBS, pH 7.5, for 15 min at378C]. Following the proteinase treatment, the sectionswere 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 sectionswere then incubated for 60 min at 378C in the TUNELreaction mixture (Boehringer Mannheim, Germany) con-sisting of 45 ml of TUNEL label (¯uorescein-dUTP anddNTP) and 5 ml of TUNEL enzyme (terminal deoxynu-cleotidyl transferase). After incubation, the sections werewashed 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 foreach step of the protocol to determine potential sources ofartifacts, namely by the use of enzymes and of methanol inacetic acid. Results showed that incubation in the enzymatic
mixture is not required, it has to be optimized for each plant

2000a, b).

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material, and should be avoided if cells or thin sections areused. Also, higher proteinase K concentrations can lead tofalse TUNEL-positive nuclei. For negative controls, onlyTUNEL label was added. Incubation of ®xed sections withDNase-I (1 mg mlÿ1, 10 min, at RT) prior to TUNELlabelling was performed for positive controls. Counter-staining for 15 min, at RT, with 1 mg mlÿ1 40-6-diamino-2-phenylindole dihydrochloride (DAPI), which speci®callybinds to adenine-thymine-rich deoxyribonucleic acids, wasperformed to distinguish non-apoptotic nuclei fromapoptotic ones. Sections were mounted in Vectashield(Vector Laboratories Inc., Burlingame, CA, USA) andobserved by ¯uorescence and/or confocal laser scanningmicroscopy (CLSM). TUNEL-positive nuclei or nucleoids¯uoresce green (excitation 450±500 nm, detection 515±565 nm), while DAPI-positive nuclei ¯uoresce blue (emis-sion 365 nm, excitation 510±530 nm). Four leaves pertreatment were randomly collected from each of the fourexperiments performed (see above) and used in TUNELassays. DAPI and TUNEL-positive cells were counted inten individual ®elds from at least six leaf sections per leaf.Experiments reported herein were repeated at least fourtimes, with all results in agreement between experiments.Results are presented as mean values+ s.d. Simple analysisof variance (ANOVA) was performed for P 5 0.05 (results

Pedroso and DurzanÐChlor

not shown).

Company, Rochester, NY, USA).

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 indistilled water or in PBS. This probe is highly speci®c forNO (Kojima et al., 1998) and has been successfully tested inplant cells (Pedroso et al., 2000a, b). After incubation, thesamples were washed twice in water, mounted in water orVectashield, and observed with a ¯uorescence or confocalmicroscope. 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 afterboiling (dead) were used as controls. For quantitation, atleast six clonal leaves were collected per treatment, andstained 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-mono-methyl-L-arginine) and hypergravity were used to investi-gate the involvement of NO on DNA fragmentation andcell death in KalanchoeÈ leaves. Leaves were incubated for3 h, at 1 g, in ®lter sterilized MS/2 medium with 10ÿ4 M

sodium nitroprusside (SNP), a NO donor; leaves incubatedin medium without SNP were used as controls. Incubationwas performed in a shaker at 60 rpm, at 23+ 28C.

In independent assays, leaves were exposed to hyper-gravity (150 g) for 3 h in ®lter sterilized MS/2 medium withand without (control) 0.5 mM NG-monomethyl-L-arginine,
L-NMMA, a competitive NO-synthase inhibitor. Exposure
in medium with 0.5 mM D-NMMA, which has nosigni®cant e�ect on NO-synthase, was performed asnegative control. Leaves kept for 3 h at 1 g under agitationwere used as an additional control. After 3 h, leaves werewashed in sterile fresh medium without SNP or NMMAand immediately assayed for NO visualization or kept inculture overnight, for the detection of apoptosis (seeabove). Double staining for simultaneous detection of NOand DNA fragmentation was also tested; live cells and leafsections were stained with DAF-2 DA, ®xed, and thenprocessed for TUNEL assay and DAPI counterstaining.This staining was possible because nuclei do not stain withDAF-2 DA; even so controls should be performed for everystep of the process. Assays in the presence of other NOdonors, NO-synthase inhibitors, NO scavengers, nitrite ornitrate supplementations were not included in the presentstudy as the results are described elsewhere (Pedroso et al.,

plasts, Nuclei and Apoptosis 985

Fluorescence and confocal microscopy

Quantitation of NO formation, DNA fragmentation andcell 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 400ASA Kodak Ektachrome ®lm. Confocal images wereobtained using Leitz Fluor 40 � and 100 � 1.3NA oilPL FLUOTAR objective lenses at a Leica TCS-NTconfocal laser scanning microscope (Leica LasertechnikGmbH, Heidelberg, Germany) equipped with Argon/Kripton and UV lasers (excitation spliter DD488/586;RSP500), and Leica TCS-NT software (TCS-NT version1.5.451). Serial confocal optical sections were taken at astep size of 1 mm. Molecular Dynamics ImageSpace soft-ware (Molecular Dynamics, Inc., Sunnyvale, CA, USA)was used to create 3-D projections of serial confocal opticalsections, merged or separated two- or three-channel 3-Dimages, and sequences of 3-D images at di�erent angles tosimulate object rotation. Images were contrast enhancedusing image processing software (Photoshop1; AdobeSystems, Inc., Mountain View, CA, USA) and printed ona Kodak ds 8650P Color Printer (Eastman Kodak

RESULTS

Detection of DNA fragmentation and cell death in di�erentgravity 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 DNAfragmentation present in leaf cells following a gravitationalforce stimuli. To determine accurately the percentage of celldeath, all cells were counterstained with DAPI afterTUNEL assay and observed in the UV and FITC channelsor, in addition, the TRITC channel. No separate UVchannel images are presented in this work. Cells with
DAPI-positive nuclei (blue; UV channel) showing no green

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¯uorescence in the FITC channel (results indicative of theabsence of DNA breaks), were considered as `non-apoptotic'. Cells with simultaneously bright greenTUNEL-positive and DAPI-negative nuclei (in the FITCand UV channels, respectively), features indicative ofirreversible DNA damage, and presenting cytologicalfeatures 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 furthercon®rmed the absence of artifacts; TUNEL was positive forboth nuclei and chloroplast nucleoids in leaf sectionstreated with DNase-I (positive controls); some nuclei werealso DAPI-positive. In negative controls (leaf sectionsincubated without TUNEL enzyme), only DAPI-positive nuclei and some DAPI-positive nucleoids were


observed. Figure 1E shows a merged confocal image of a

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non-apoptotic, double stained mesophyll cell. Most chlor-oplasts, showing starch grains and bright red auto¯uores-cence (chlorophyll), were distributed close to the cell wall,


far from the nucleus (Fig. 1E).

(Fig. 1K), with or without the presence of nuclear bodies.

Cell death in leaves is di�erentially a�ected by di�erentgravity environments

Culture in 2 � 10ÿ4 g (clinorotation; simulated hypo-gravity), 1 g (Earth's gravity; controls) and 20 and 150 g(hypergravity), in darkness or a 16 h photoperiod, showedthat the gravity environment signi®cantly a�ects DNAfragmentation and cell death in K. daigremontiana leaves,for both plantlets and isolated leaves. In this report weemphasize the results obtained with isolated leaves.

Figures 1A±C and 2 show that the percentage of leaf cellsexhibiting irreversible DNA fragmentation (TUNEL-positive cells) increased proportionally with the increaseof gravitational force, from 2 � 10ÿ4 g to 150 g. Labelledcells were detected in leaf epidermis (mainly guard cells),parenchyma and tracheary elements undergoing terminaldi�erentiation (Fig. 1A±D). Guard cells (Fig. 1F) andsubepidermal parenchyma cells (Fig. 1H) were the mosta�ected by gravity changes. Leaf culture in darkness, inhypogravity or 1 g, increased the number of TUNEL-positive cells. However, di�erences between leaf culture indarkness or light were not signi®cant (P 5 0.05) (Fig. 2).

Except for later stages of cell death, the pattern ofnuclear degeneration was identical for clinorotated, controland centrifuged cells. Early stages of apoptosis werecharacterized by bright green TUNEL-positive nuclei withthe same shape as DAPI-positive ones (Fig. 1D and F).Disorganization of the nuclear envelope and marginaliza-tion of chromatin were also observed in subsequent stagesof the process (Fig. 1H and I), and con®rmed bytransmission electron microscopy (data not shown). At1 g, bright areas of labelled DNA fragments were
frequently concentrated at the periphery of the nucleus,
FIG. 1. In situ detection of nuclear and chloroplastidial DNA fragmentatiFluorescence microscope images of leaf sections obtained using a FITC ®detect DNA fragmentation (green ¯uorescence). TUNEL-positive nucleicollected and processed for TUNEL (see Materials and Methods) after 1observed between treatments were identical for di�erent focal planeshypergravity, 20 g. Bar � 20 mm. D±O, Confocal laser scanning microsccounterstaining (UV channel); red, when present, is due to chlorophyll autnuclei (green) in a cross-section from a leaf exposed to 20 g, for 15 d (merleaf parenchyma cell from a leaf cultured in 1 g, showing a DAPI-positive(3-D projection; UV-FITC-TRICT). Bar � 5 mm. F, Apoptotic guard cspots) at 20 g (merged image, UV-FITC channels only to eliminate chloroin an epidermal cell. Note the presence of nuclear bodies (bright spotsBar � 3.0 mm. H, Nuclear and chloroplastidial DNA fragmentation in meTRITC). Bar � 11 mm. I, Pseudo-colour image of an apoptotic nucleus froHot colours correspond to areas of high DNA fragmentation (FITC chaExample of an apoptotic nucleus from a cell of a leaf cultured in hypergranucleus (UV-FITC-TRITC). Bar � 1.7 mm. K, Example of an apoptotic nnuclear fragmentation and presence of nuclear bodies (UV-FITC-TRIchloroplasts of mesophyll cells after 15 d of leaf exposure to hypergravitpositive cell from a leaf exposed to hypergravity (3-D projection; UV-FITCof chloroplasts around the nucleus and the presence of a network ofTUNEL-positive chloroplasts concentrate around the nucleus or irradiateBar � 31 mm. O, This image shows the intricate network of connections

Yellow lines were drawn over the 3-D p

while dark areas of fully degraded nuclear materialappeared in the nucleus core (Fig. 1I). TUNEL-positivechloroplasts, close to the nucleus, with one to seven brightgreen ¯uorescent spots (indicative of nucleoid DNAfragmentation) were always observed in apoptotic guardcells (Fig. 1F), and in parenchyma cells undergoingapoptosis (Fig. 1H and L).

In contrast to apoptotic cells in 1 g (Fig. 1I), theapoptotic cells exposed to hypergravity presented drasticnuclei condensation and blebbing, resulting in nucleifrequently surrounded by bright green ¯uorescent bodies(Fig. 1G and J). 3-D projections of serial confocal opticalsections from images identical to those on Fig. 1G and Jshowed several stages of bleb formation on the nuclearsurface (image not shown). TUNEL-positive globularbodies were observed close to the apoptotic nucleus, bothin hypergravity- and hypogravity-exposed leaf cells (Fig. 1Jand K). Figure 1J shows those bodies detaching andmoving away from a condensed nucleus. In epidermalcells, the migration of those nuclear bodies to the peripheryof the cell, forming `lines' of bright spots, was frequentlyobserved (Fig. 1G). Transmission electron images con-®rmed the nuclear blebbing observed and formation ofvesicles containing nuclear materials (images not shown).Degeneration of the nuclear envelope and of mitochondriaaround the nucleus was also common. In hypergravity, theappearance of a second nucleolus before nuclear conden-sation was also observed (image not shown).

In simulated hypogravity, nucleus fragmentation in twoportions was commonly observed for elongated nuclei


Di�erential TUNEL reaction with chloroplast di�erentiationand 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
on and cell death (apoptosis) in KalanchoeÈ daigremontiana leaves. A±C,lter (excitation 450±490 nm; emission 520 nm) after TUNEL assay toare in green; red is due to chlorophyll auto¯uorescence. Leaves were5 d exposure to di�erent levels of gravity stimulation. The di�erences. A, Hypogravity, 2 � 10ÿ4 g; B, Earth's gravity, 1 g (control); C,opy images acquired after TUNEL assay (FITC channel) and DAPIo¯uorescence (TRITC channel). D, DAPI- (blue) and TUNEL-positiveged image; UV-FITC). Bar � 11 mm. E, Non-apoptotic, double stainednucleus and TUNEL-negative chloroplasts (red auto¯uorescence only)ells showing nuclear and chloroplastidial DNA fragmentation (greenphyll auto¯uorescence). Bar � 3.5 mm. G, Nuclear DNA fragmentation) migrating towards the cell wall at 20 g (merged image, UV-FITC).sophyll cells after 15 d of exposure to 150 g (3-D projection; UV-FITC-m a leaf cell cultured in 1 g. Note the marginalization of the chromatin.nnel; no ¯uorescence on UV and TRICT channels). Bar � 1.2 mm. J,vity. Note the presence of nuclear bodies detaching from the condenseducleus from a cell of a leaf cultured in simulated hypogravity. Note theTC). Bar � 1.6 mm. L, TUNEL-positive nucleoids (green spots) iny (3-D projection; UV-FITC-TRITC). Bar � 2.2 mm. M±O, TUNEL-). Images arti®cally coloured to enhance contrast. M, Note distributionconnections between chloroplasts (arrows). Bar � 6.8 mm. N, Linkedtoward the cell wall, as shown by the red lines drawn over the image.(yellow lines) observed between labelled and unlabelled chloroplasts.rojection series in M. Bar � 26 mm.

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FIG. 2. Cell death (TUNEL-positive cells) in the epidermis andmesophyll of KalanchoeÈ leaves after 15 d exposure to di�erent levels ofgravity stimulation (2 � 10ÿ4, 1, 20 and 150 g) and light conditions(darkness and a 16 h photoperiod). Leaves were ®xed immediately aftereach treatment, sectioned, processed for TUNEL and counterstainedwith DAPI. Leaves cultured at 1 g, in darkness and light, were used ascontrols. Leaf sections treated with DNase-I after ®xation andpermeabilization (positive control) displayed 85% TUNEL-positivecells. Leaf sections processed without addition of terminal transferasedid not label. DAPI-positive vs. TUNEL-positive cells in the epidermis(h) and mesophyll (j) were counted in ten individual microscope ®eldsfrom at least six leaf sections from each of the four leaves randomlycollected per treatment per experiment. Values are the means of four 0+24 h 10+24 h 30+24 h 60+24 h

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FIG. 3. Quantitation of nitric oxide (NO) formation and of nuclearand chloroplast DNA fragmentation following acute hypergravitytreatments. A, Leaves, collected at the times indicated after hyper-gravity exposure (150 g; hyper-G), were sectioned, stained for 1 h with10 mM DAF-2 DA for nitric oxide visualization, ®xed, processed forTUNEL and counterstained with DAPI. Leaf sections, fresh and ®xed,not assayed for TUNEL, treated with DNase-I, processed withoutterminal transferase, and without DAPI staining, were used as controlsof this staining assay. B, Hyper-G treated leaves, kept for 24 h in 1 gunder a 16 h photoperiod, were processed as described for A. Leavesnot exposed to hypergravity (0 � 24 h) were also collected and used ascontrols. Three to ®ve leaf sections (90 mm), performed in at least sixclonal leaves per treatment, were used to quantify the percentage of leafcells with NO (shaded line) and, within TUNEL-positive cells, thosepresenting nucleoid (m) and nuclear (j) DNA fragmentation. Valuesare the mean of six independent experiments. Error bars are lower

than 7%.

988 Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis

Fig. 1E); (2) orange, with starch grains, TUNEL-positive(Fig. 1H and L); and (3) bright orange ¯uorescence at theperiphery, almost completely ®lled with starch, TUNEL-negative.

The number of labelled chloroplasts increased withincreasing gravitational force. Labelled chloroplasts werealways present when nuclei presented severe DNA frag-mentation (Fig. 1H and L). However, at lower gravitationalforces, including 20 g, subepidermal parenchyma cellspresenting only labelled chloroplasts were frequentlyobserved (image not shown).

3-D projections from sequence series of optical sections(Fig. 1M±O) showed the presence of straight connectionsamong chloroplasts of apoptotic cells (Fig. 1M), notobserved in non-apoptotic cells. Labelled chloroplastswere concentrated around the nucleus and close to thecell wall. Distribution of chloroplasts around the nucleuswas not observed in non-apoptotic cells. In Fig. 1N, redlines were drawn over the projection series of opticalsections to show the TUNEL-positive chloroplasts con-nected with each other, from the cell wall to the nucleus.3-D imaging analysis further revealed an intricate networkof connections between labelled chloroplasts and TUNEL-

independent experiments. Error bars are lower than 8%.

negative ones (Fig. 1O).

Nuclear DNA fragmentation is preceded by nucleoid DNAfragmentation

In order to determine how fast leaves responded tochanges of gravity, in situ detection of DNA fragmentationwas performed in clonal leaves immediately (0 h) and 24 hafter 10, 30 and 60 min at 150 g. Ten minutes' (T10)
exposure to 150 g drastically increased the number of
TUNEL-positive cells. This increase was entirely due tonucleoid DNA fragmentation, as the number of cells withnuclei presenting DNA fragmentation was identical to 1 gcontrols (Fig. 3A). Guard cells and subepidermal paren-chyma cells were the most a�ected. Longer exposures, T30and T60 min, further increased nucleoid DNA fragmenta-tion (Fig. 3A). DNA fragmentation in brightly DAPI-stained nuclei also increased, compared to T0 and T10 min,mainly in epidermal cells (Fig. 3A).

Detection of DNA fragmentation 24 h after leafexposure to 150 g showed that the number of TUNEL-positive nuclei and the extent of nuclear DNA fragmenta-tion had further increased compared to the previous day(Fig. 3B). The nuclei were no longer DAPI-positive, thenumber of cells with TUNEL-positive nucleoids decreasedsigni®cantly (Fig. 3B), and death cells were detected in all
samples. The highest number of labelled chloroplasts and

FIG. 4. Visualization of nitric oxide (NO) and DNA fragmentation in K. daigremontiana leaves exposed to hypergravity. A, Presence of NO inepidermal 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 � 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 � 3.4 mm. C, Fluorescence microscope image of a mesophyllsection of a centrifuged leaf stained for NO as described in A. Note the presence of NO (green) in some chloroplasts while others show no NOproduction (star). Bar � 12.5 mm. D, Magni®ed confocal image (merged FITC-TRITC) of C. DAF-2 DA-positive chloroplasts in green.Bar � 2 mm. E, Simultaneous detection of NO and DNA fragmentation in a mesophyll cell by DAF-2 DA and TUNEL, respectively. This waspossible 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 thenucleus are not stained (circular shadows). Nuclear DNA fragmentation is detected, although the nucleus was still DAPI-positive (image notshown). 3-D projection, only the FITC channel is shown. Bar � 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 � 3.6 mm. G, TRITC-transmission. Bar � 3.6 mm. H, Merged confocal image showing the connections between DAF-2 DA-positive andnegative chloroplasts (asterisk). This image suggests that NO di�usion might occur between chloroplasts. Yellow results from the merging of

FITC-TRITC channels. Bar � 2.8 mm.

Pedroso and DurzanÐChloroplasts, Nuclei and Apoptosis 989

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 DNAfragmentation

DAF-2 DA staining (green) of fresh leaves immediatelyand 24 h after 10, 30 and 60 min in hypergravity (150 g),showed that NO formation signi®cantly increased afterexposure, decreasing to levels higher than those detected forcontrols 24 h later (Fig. 3A and B). NO was detecteddi�used in the cytosol of epidermal cells, mainly in guardcells (Fig. 4A and B), or it was just detected in chloroplasts(Fig. 4C and D). Sectioned and stained controls (leaves in1 g for the same period of time) con®rmed that the NOburst detected was a consequence of hypergravity exposureand not of sample manipulation.

To investigate whether NO formation was involved inDNA 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), aNO-synthase inhibitor. Incubation in hypergravity inmedium with D-NMMA, which has no signi®cant e�ecton NO-synthase, was performed as negative control. Leavescollected immediately after the treatments were sectioned,stained with DAF-2 DA, processed for TUNEL, and thencounterstained with DAPI. Figure 5 shows the resultsobtained immediately after treatment. SNP and hyper-gravity both increased the number of cells showing NO andDNA fragmentation, compared to 1 g controls (Fig. 5).Culture in hypergravity with L-NMMA signi®cantlydecreased NO formation and DNA fragmentation, inboth epidermal and mesophyll cells, compared to centri-fuged samples without the inhibitor, and to 1 g controls(Fig. 5). NO formation and DNA fragmentation wasidentical for samples cultured in hypergravity with andwithout D-NMMA (data not shown), recon®rming theinhibitory e�ect of L-NMMA. These results showed thatNO formation was directly correlated with DNA fragmen-tation. Additional assays in which leaves were ®xed and
processed for TUNEL without previous in vivo incubation

not shown).

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for 1 h in DAF-2 DA, showed that nucleoid DNAfragmentation increased during the ®rst hour immediatelyafter exposure (data not shown). Figure 5 shows that theinhibition of NO formation by L-NMMA was moree�ective in chloroplasts (empty bars; mesophyll cells) thanin the cytosol (shaded bars; epidermal cells).

3-D projections showed that chloroplasts brightly stainedfor NO were concentrated close to the cell wall (Fig. 4E),whereas dimly stained chloroplasts converged to thenucleus. The chloroplasts next to the nucleus were notstained (Fig. 4E, dark shadows on the nucleus). At thatmoment (1 h after the 3 h treatment), DNA fragmentationwas already detected in the nucleus (Fig. 4E), althoughDAPI ¯uorescence was still bright in the UV channel(image not shown).

NO was detected di�used in the stroma of the DAF-2DA-positive chloroplasts preferentially around starchgrains (Fig. 4F±H). The red auto¯uorescence of thechlorophyll frequently surrounded, completely or partially,the green ¯uorescent core (Fig. 4F and G). The appearanceof a yellow colour after merging the confocal images fromthe FITC and TRITC channels con®rmed that NO couldalso be detected close to the thylakoid membranes(Fig. 4H).

DAF-2 DA-positive connections between stained-stainedand stained-unstained chloroplasts were also observed(Fig. 4H). However, in the latter, the side of the tubularconnection in contact with the unstained chloroplast wasnot ¯uorescent, suggesting the possibility of NO di�usionbetween chloroplasts.

Identical results were obtained for the other hypergravity


treatments. As for the TUNEL assay, it was the epidermal

0 10 20 30 40 50 60 70 80 90 100

0 5 10 15 20 25 30 35 40 45

Hyper+L-NMMA

Hyper-G

10-4 SNP

Control

Percent of NO stained cells

Percent of TUNEL-positive cells

FIG. 5. Induction or inhibition of nitric oxide (NO) formation andDNA fragmentation by chemical agents and centrifugation. KalanchoeÈleaves were incubated for 3 h in 1 g in culture medium with or without10ÿ4 sodium nitroprusside (SNP), a nitric oxide donor, or at 150 g,with or without NG-mono-methyl-arginine (NMMA), a NO-synthaseinhibitor, to determine the e�ect of NO on DNA fragmentation.Leaves were sectioned immediately after each treatment, stained for 1 hwith 10 mM DAF-2 DA for nitric oxide visualization, ®xed, processedfor TUNEL to detect DNA fragmentation and counterstained withDAPI. TUNEL-positive cells (j; epidermis and mesophyll) and DAF-2 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 asdescribed 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 NO-positive cells were/became apoptotic. In all assays, leaf-cellresponse was identical for both leaves and plantlets. Inboth, leaf-plantlet formation paralleled the cell deathincrease with the increase of gravitational force (results


DISCUSSION

The present results provide microscopic and TUNELstaining evidence that cell death is induced in response togravity changes. Cell death increased with increased levelsof gravity stimulation, and was shown to be increased orreduced by chemical agents (SNP or L-NMMA, respect-ively). Plantlet and leaf culture for 15 d under multiples ofEarth's gravity was expected to induce a stress response inleaf cells, which could eventually lead to cell death. Theseresults are in accordance with previous reports showing thatcentrifugation and other forms of mechanical stress canlead to cell damage and/or cell death (Pedroso and Durzan,1998, 1999; Magalhaes et al., 1999). Cell death wasincreased by exposure to a NO generator (SNP) andreduced by a NO-synthase inhibitor (L-NMMA). Additionof D-NMMA did not a�ect cell death. This indicates thatthe cell death process induced was under physiologicalcontrol and, possibly, mediated by NO.

In both hyper- and hypogravity, cell death was charac-terized by distinct chromatin marginalization, nuclearcondensation and blebbing, resulting in the formation ofnuclear-apoptotic bodies. These are typical features ofapoptosis in animal (Bowen et al., 1996) and plant cells(Havel and Durzan, 1996a; Wang et al., 1996; Yeng andYang, 1998). In simulated hypogravity, nuclei often brokeinto two fragments (see Fig. 1K), a phenomenon notobserved in cell death induced by hypergravity (seeFig. 1J). We did not determine the cause of this di�erence.Nuclear breakage after formation of small nuclear-apopto-tic bodies might represent a later stage of nucleardegeneration. Based on the above, these results providestrong evidence that the cell death process induced inKalanchoeÈ leaves was apoptosis.

The in vivo visualization of NO by DAF-2 DA requiredan incubation period of 1 h, whereas determinations ofDNA fragmentation were made on destructively sampledleaves. Nevertheless, using acute hypergravity treatments(10±60 min) and several experimental controls, it waspossible to show that the NO bursts were caused byexposure to hypergravity and not by sample manipulationand/or sectioning, and that they preceded chloroplast DNAfragmentation. Chloroplasts were the ®rst visible organellesto show NO and DNA fragmentation. K. daigremontianachloroplasts have small, uniformly dispersed nucleoids inthe matrix between the thylakoid membranes and/or thegrana stalks (Kuroiwa et al., 1981). Treatments with SNPand L-NMMA enabled us to show a direct correlationbetween NO and DNA fragmentation in chloroplasts and
in tissues (epidermis and mesophyll). Identical results were

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recently reported for Arabidopsis thaliana (Garces et al.,1999) and Taxus brevifolia (Pedroso et al., 2000b). NoDAF-2-positive cells or DNA fragmentation were observedwhen centrifugation was performed in the presence ofcarboxy-PTIO, a NO scavenger (Pedroso and Garces, pers.comm.). The consistent staining of KalanchoeÈ chloroplastswith DAF-2 DA suggests that NO was being produced inthese organelles, and that NO was directly or indirectlyresponsible for the damage of chloroplast DNA. Thisreinforces previous results on the possible involvement offree radicals in signal pathways leading to PCD (Jacobsen,1996; Panavas and Rubinstein, 1998), and supports theinvolvement of NO in DNA damage and plant cell death,as described for animal systems (Wink et al., 1991; BruÈ ne


et al., 1998; Tannenbaum, 1998).

Chloroplast DNA fragmentation was detected 10 minafter exposure to hypergravity. Nuclear DNA fragmenta-tion was ®rst detected 20 min later (at 30 min of exposure),increasing until 24 h after the treatments, when chloroplastdegeneration was severe. In our study, at 30 and 60 min ofexposure, the nuclei that labelled for TUNEL were alsobrightly stained for DAPI. This indicates that most of theirDNA was still intact. After 24 h, no intact DNA wasdetected on TUNEL-positive nuclei. Morphologicalchanges occurring in chloroplasts before nuclear changeswere also reported for resistant tobacco plants infected withTMV (Mittler et al., 1996), and during xylogenesis inZinnia cells (Fukuda et al., 1998). Our results are also inaccordance with those described for PCD in trachearyelements, a process estimated to be completed in 8 to 12 h(Groover and Jones, 1999), and for toxin-induced cell deathin asc/asc tomato protoplasts (6 to 16 h) (Wang et al.,1996).

In mesophyll cells, environmental stresses are readilyperceived by chloroplasts and elicit an oxidative response(NoodeÂ n, 1988; Allen, 1993; BruÈ ggemann et al., 1994;Green and Fluhr, 1995; Allen and Raven, 1996; Allan andFluhr, 1997; Buchanan-Wollaston, 1997). NO reacts withsuperoxide (Oÿ2 ) to form peroxynitrite (OONOÿ) (Pryorand Squadrito, 1995), which damages DNA, RNA,proteins and lipids (Stamler et al., 1992). Thus it is notsurprising that chloroplast-containing cells closest to theregion of stimuli/stress application (guard cells andsubepidermal parenchyma cells) were the most sensitive toexternal changes (light or gravity). The increase in cell deathrecorded in darkness was also probably related to


senescence in photosynthetic tissues (Panavas and

FIG. 6. Summary of the time sequence of events leading to cell death inKalanchoeÈ mesophyll cells as a response to a change in gravity. A, Leafexposure to higher levels of gravity stimulation (hypergravity) induces aNO burst (green) in the chloroplasts (Ch; red) of mesophyll cells thatare localized close to the cell wall (Cw), on the site of force application.The NO burst precedes and correlates with nucleoid DNA fragmenta-tion during the following 3 h. No nuclear DNA fragmentation isdetected (blue; DAPI). A bright red auto¯uorescence from chlorophyllis present. B, After 4 h, the number of chloroplasts stained for NO isstill high, nucleoid DNA fragmentation is highest and nuclear DNAfragmentation is detected in DAPI-positive nuclei (DAPI-TUNEL).Chloroplasts redistribute. Unstained chloroplasts move toward thenucleus. In the following hours, the auto¯uorescence of TUNEL-positive chloroplasts close to the cell wall becomes orange, indicative ofa reduction of chlorophyll content, and a network of connectionsbetween chloroplasts becomes apparent. C, After 9±12 h, the numberof chloroplasts stained for NO is reduced and microscopic evidence ofsevere chloroplast degeneration is present. TUNEL-positive chloro-plasts, when present, are localized close to apoptotic nuclei. Anintricate network of connections between chloroplasts is observed.Nuclear condensation and blebbing with formation of apoptotic bodiesis detected. The time required for completion of B and C varies withthe intensity and duration of the stimulus. Cytological evidenceindicates that the process of cell death by apoptosis can be completedin 9 h. S and r represent `turning points' in the process, where S arecells that survive and adapt to the stimulus applied and proliferate,while r represents cells that through addition of L-NMMA could berescued from phase A and prevented from dying. This suggests theexistence of at least a checkpoint (*) between A and B, and indicatesthat the process can be manipulated within the ®rst hours following the

application of the stressful stimulus.

and cell death by apoptosis.

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Rubinstein, 1998). A direct consequence of the exposure toincreasing levels of gravity stimulation was the increase andspread of the population of TUNEL-positive parenchymacells to deeper cell layers.

Because nitrate was used in the culture medium, wecannot exclude the conversion of nitrite to NOx(NO � NO2) by nitrate reductases, and/or non-enzymaticones, resulting from chemical reactions between plantmetabolites and accumulated NOÿ2 and/or decompositionof 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 stainingaround the starch grains (see Fig. 4F) might indicate non-enzymatic NO generation (Nishimura et al., 1986). How-ever, the inhibitory e�ect of L-NMMA on NO production(see Fig. 5) led us to reject this possibility. Our resultssuggest the presence of an inducible NO-synthase inKalanchoeÈ leaf cells. The recent report of a gene inArabidopsis thaliana with sequence similarity with animalNO-synthase (Mayer et al., 1999), further supports thisassumption. The localization of the NO-staining, in thecytosol and/or chloroplasts coincides with sites of knownlocation of soluble L-arginine (Ludwig, 1993), and thosepreviously reported in in situ localization assays (Barrosoet al., 1999; Pedroso et al., 2000a, b).

The detection of NO and DNA breaks in chloroplastswas accompanied by a decrease in chlorophyll auto¯uor-escence, increase in starch content, and followed by severenuclear DNA damage. Chloroplast degeneration wascon®rmed by electron microscopy and by a concomitantloss of photosynthetic capacity (unpubl. res.). Membranelipids of chloroplasts could be one of the primary targetsfor NO attack, detrimentally a�ecting the e�ciency ofphotosynthesis (Leshem et al., 1997). The di�erentialTUNEL reaction with chloroplast di�erentiation andgravitational forces was probably due to the `redox state'of the plastid at the moment of stimuli/stress exposure andits ability to cope with an additional stress (Allen andRaven, 1996).

TUNEL-positive chloroplasts were linked by cytoplasmicconnections and concentrated around the nucleus (seeFig. 1M). Linkages irradiated in lines to the cell wall (seeFig. 1N). Exchange of protein molecules through connec-tions between chloroplasts was recently demonstrated(KoÈ hler et al., 1997; Shiina et al., 2000). Confocal imagesfrom parenchyma cells double stained with DAF-2 DA-TUNEL (see Fig. 4E), suggested that NO di�used along acytoplasmic network to the nucleus where endonucleaseactivity was then initiated.

How the chloroplast stress responses leading to cell deathwere related to the network of connections remains unclear.This intricate network of connections was similar to acortical endoplasmic reticulum (see Fig. 1M) (Gunning andSteer, 1996). The network could engulf NO-producingchloroplasts to prevent further cellular DNA damage, andpromote calcium in¯ux favouring DNA damage andchloroplast elimination. It could also increase moleculetransport among degenerating organelles and the neigh-bouring cells, prior to cell death. Information transfer to


the nucleus, by direct chloroplast-nucleus or chloroplast-

nucleolus interaction, or by the release of signallingmolecules like octadecanoid 12-oxo-phytodienic acid(Stelmach et al., 1998), could trigger silent gene-activationleading to cell adaptation and survival ( for reviews seeForsburg and Guarente, 1989; Oelmuller, 1989; Taylor,1989; Zachleder et al., 1989, 1995; Ehara et al., 1990; Susekand Chory, 1992; Susek et al., 1993; Gillham, 1994).Cellular degradation products from apoptosis (Havel andDurzan, 1996a, b) would be salvaged to sustain adaptation,viability, and ontogeny of the neighbouring cells, assuringthe survival of the species in a harsh(er) environment.Speci®c checkpoints that regulate plant cell cycle pro-gression in response to oxidative stress (Reichheld et al.,1999), could explain why some cells undergo programmedcell death, while others undergo division leading to leaf-plantlet formation. NO could also play a role in such asituation depending on its concentration and reactivity withsuperoxide concentration (Shen et al., 1998).

At the organ level (leaf), the increases/decreases in NOformation and cell death were paralleled by increases/decreases in leaf-plantlet formation (data not shown;Pedroso and Durzan, 1998). Thus, the involvement of NOin DNA damage in KalanchoeÈ cells does not exclude apossible role of NO as a regulator of leaf senescence, assuggested by Leshem et al. (1998). Senescence leads to adecrease in chlorophyll content, chloroplast degeneration(including nucleoid DNA fragmentation) and, ultimately,nuclear condensation and internucleosomal DNA frag-mentation ( for a review see Hensel et al., 1993; Smart, 1994;Humbeck et al., 1996; Bleecker and Patterson, 1997;Buchanan-Wollaston, 1997; OrzaÂ ez and Granell, 1997;Gray and Johal, 1998), exhibiting cytological features ofPCD by apoptosis (Yeng and Yang, 1998). Our resultssuggest that KalanchoeÈ leaves responded to a gravity changeby forming NO, which triggered a senescence-like processthat exhibited characteristics of apoptosis. Simulatedgravity from 2 � 10ÿ4 to 150 g, increased NO-related celldeath (Fig. 6). In vitro cultured leaves of K. daigremontianaprovide a unique experimental system for studying the e�ectof environmental stresses (gravity or other) (Resende andPereira-da-Silva, 1965; Pedroso and Durzan, 1999), on apotential homeostatic balance between plantlet formationand apoptosis in this species.

In conclusion, cell death in KalanchoeÈ leaves increasesproportionally with the increase of gravitational force, from2 � 10ÿ4 to 150 g. Stressful gravity environments lead to aNO burst in chloroplasts. In some cells this triggers asequence of events that leads to chloroplast DNA damage,chloroplast degeneration, followed by nuclear degeneration


ACKNOWLEDGEMENTS

This work was supported by FundacË aÄ o para a CieÃ ncia e aTecnologia (contract PRAXIS XXI 3/3.1/CTAE/1930/95),INVOTAN (3/B/96/PO) and Centro de Biotecnologia
Vegetal (Lisboa, Portugal).

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Effect of Different Gravity Environments on DNA

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