Planta (2002) 214: 608615
DOI 10.1007/s004250100646
O R I GI N A L A R T IC L E
S. Munne-Bosch L. Alegre
Plant aging increases oxidative stress in chloroplasts
Received: 30 April 2001 / Accepted: 12 July 2001 / Published online: 7 September 2001
Springer-Verlag 2001
Abstract Aging has received considerable attention in
biomedicine, but little is known about the regulatory
mechanisms responsible for the aging not associated
with senescence in plants. This study provides new in-
sights into the relationship between oxidative stress and
plant aging, and points out chloroplasts as one of the
target organelles of age-associated oxidative stress in
plants. We simultaneously analyzed lipid oxidation,
photosynthesis, chlorophyll content, de-epoxidation
state of the xanthophyll cycle, and levels of chloroplastic
antioxidant defenses such as b-carotene and a-tocoph-
erol in leaves of the same age in 1-, 3- and 7-year-old
Cistus clusii Dunal plants growing under Mediterranean
eld conditions. Enhanced formation of malondialde-
hyde in leaves (2.7-fold) and chloroplasts (2.8-fold),
decreased photosynthetic activity (25%), and lower
chlorophyll (ca. 20%) and chloroplastic antioxidant
defense levels (ca. 25%85%) were observed in 7-year-
old plants, when compared with 1- and 3-year-old
plants. The dierences observed, which were associated
with plant aging, were only noticeable in mature non-
senescing plants (7-year-old plants). No dierences were
observed between pre-reproductive (1-year-old plants)
and young plants (3-year-old plants). This study shows
that from a certain age, oxidative stress increases
progressively in chloroplasts as plants age, whereas
photosynthesis is reduced. The results indicate that the
oxidative stress associated with the aging in plants
accumulates progressively in chloroplasts, and that the
contribution of oxidative stress to aging increases as
plants age.
Keywords Aging Antioxidant Chloroplast Cistus
clusii Oxidative stress Xanthophyll cycle
S. Munne-Bosch L. Alegre (&)
Departament de Biologia Vegetal,
Facultat de Biologia, Universitat de Barcelona,
Av. Diagonal 645, 08028 Barcelona, Spain
E-mail: leonor@porthos.bio.ub.es
Fax: +34-93-4112842
Abbreviations DPS: de-epoxidation state of the
xanthophyll cycle /PSII: relative eciency of photo-
system II photochemistry MDA: malondialdehyde
PPFD: photosynthetic photon ux density RWC:
relative water content TBA: thiobarbituric acid
Introduction
In plant development, pre-reproductive plants are
considered juveniles; the onset of owering and seed
production marks a transition phase, and fully repro-
ductive plants are considered mature (Bond 2000).
Plants maintain the capacity to develop new leaves and
grow throughout their life until they enter a develop-
mentally controlled senescing process that inevitably
leads to plant death. Aging has been classically dened
as the accumulation of changes in plant development
responsible for slow, progressive, and sequential altera-
tions that accompany the plants as they age (Harman
1981, 1991).
In animals, age-associated disorders in the cell are
believed to be connected with the time-dependent shift in
the antioxidant/pro-oxidant balance in favor of oxida-
tive stress, and it has been suggested the mitochondria
are the target organelles for oxidative stress in age-
associated changes observed in animals (Ashok and Ali
1999; Rustin et al. 2000). The role of oxidative stress in
plant senescence (Smart 1994; Nooden and Guiamet
1996; Buchanan-Wollaston 1997; Dangl et al. 2000;
Quirino et al. 2000) and in the response of plants to a
number of environmental stresses (Smirno 1993; Foyer
et al. 1994; Asada 1999) has been demonstrated. How-
ever, the role of oxidative stress and antioxidants in the
aging of plants before senescence occurs is poorly
understood, as is, to an even greater extent, which
organelles are responsible for mediating age-associated
changes in plants.
Some studies have focused on the study of chloro-
plast aging in vitro, i.e. the changes in chloroplast
morphology, photosynthesis or photosynthetic pigments

as isolated chloroplasts age in controlled conditions
(Srichandan et al. 1989; Pancaldi et al. 1996), or leaf
aging, i.e. the changes in photosynthesis, photosynthetic
pigments or antioxidants as leaves age (Murchie et al.
1999; Havaux et al. 2000). At the whole-plant level, it
has been shown that photosynthesis generally decreases
in leaves as plants age (Bond 2000, and references
therein). However, the roles of oxidative stress and
chloroplastic antioxidant defenses in aging in non-
senescing plants have not been examined so far. Here,
we investigate the role of chloroplasts as targets of the
oxidative stress associated with aging in plants.
Materials and methods
Plant material and growth conditions
Cistus clusii Dunal is an evergreen half-shrub typical of the
Mediterranean region, and has a life span of about 1520 years.
The study was conducted using three groups of plants of dierent
ages: (i) 1-year-old plants, which can be considered juveniles be-
cause they were in a pre-reproductive stage; (ii) 3-year-old plants,
which can be considered young or in the transition phase (the
formation of owers was observed once); and (iii) 7-year-old
plants, which can be considered mature non-senescing plants (the
formation of owers was observed several times and the plants
continued owering; Bond 2000).
The plant material was obtained as follows. C. clusii seedlings
were grown in 0.5-l pots containing a mixture of soil:peat:perlite
(1:1:1, by vol.). Plants were maintained in a greenhouse with a
controlled temperature (24 C/18 C, day/night) and were watered
twice a week, once with tap water and once with Hoagland's
solution. Three groups of 1-year-old plants were transplanted to
the experimental elds of the University of Barcelona. The exper-
imental area consisted of three plots of 4.5 m2 each of calcic
Luvisol (FAO) homogenized articially 10 years ago. Before the
plants were transferred, the soil was ploughed and treated with
N:P:K (1:1:1) fertilizer at the rate of 100 kg ha1, and an analysis of
the mineral composition of the soil was carried out annually to
avoid mineral deciency in the plants. Plants were transplanted
during the autumns of 1993, 1997 and 1999, and 16 plants per plot
were distributed homogeneously in a square, 1 m apart, so all
plants had the same orientation to the sun and there were no dif-
ferences in leaf temperature between the dierent groups of plants
during the experiments.
Lipid oxidation in leaves and chloroplasts, photosynthetic
activity, chlorophylls, xanthophyll cycle, chloroplastic antioxidant
(b-carotene and a-tocopherol) levels, and water relations were
analyzed simultaneously in the three age groups from January to
July 2000. Sixteen 1-year-old, eight 3-year-old, and eight 7-year-old
plants, of approximately the same size within each group, were
chosen for the measurements. Leaves of the same ontogeny (fully
expanded young leaves situated at 515 cm from the apex) from
each group of plants were selected for all the measurements. The
leaves selected for the measurements appeared in the autumn of
1999 as re-sprouts, and were of the same age in the three age groups
of plants.
Plants received water exclusively from rainfall during the
growth and study period. Environmental conditions were moni-
tored at 5-min intervals throughout the day with a weather station
(Delta-T Devices, Newmarket, UK). The photosynthetic photon
ux density (PPFD) was measured with a Quantum Sensor
(Li-Cor, Lincoln, Neb., USA). Air temperature and relative hu-
midity were measured with a thermohygrometer (Vaisala, Helsinki,
Finland). The vapor-pressure decit was determined from relative
humidity and air temperature data following Nobel (1991).
Precipitation was measured with a standard rain gauge.
609
Estimation of lipid oxidation
The extent of lipid oxidation was estimated by measuring the
amount of malondialdehyde (MDA) in leaves and chloroplasts by
the method described by Hodges et al. (1999), which takes into
account the possible inuence of interfering compounds in the
assay for thiobarbituric acid (TBA)-reactive substances. For ana-
lyses of MDA in leaves, leaves were collected at predawn (1 h be-
fore sunrise), midday (at maximum incident PPFD) and evening
(1 h after sunset), immediately frozen in liquid nitrogen and stored
at 80 C until analysis. For analyses of MDA in chloroplasts,
leaves were collected at midday, and were immediately subjected to
cell fractionation as described by Munne-Bosch and Alegre (2001).
After grinding the sample in isolation buer [0.51.5 M sorbitol
(depending on the plant water status), 50 mM Tricine, 1 mM
dithiothreitol, 1 mM MgCl2, 1 mM citric acid, 1 mM butylated
hydroxytoluene (BHT), 0.1% (w/v) bovine serum albumin, pH 7.8],
the homogenate was ltered through four layers of cheesecloth and
centrifuged at 2,500 g for 4 min. The pellet was resuspended in
isolation buer and then centrifuged at 200 g for 1 min. The
chloroplasts in the supernatant were sedimented by centrifugation
at 2,500 g for 4 min. Chloroplasts were puried by resuspending
the pellets in isolation buer, layering onto 10 ml of 25% (by vol.)
Percoll (in isolation buer), and centrifuging at 15,800 g for
20 min. The chloroplast pellets were resuspended in isolation buf-
fer, centrifuged at 2,500 g for 4 min, and used immediately. The
identity and purity of chloroplasts were determined by assaying
amounts and activities of appropriate markers, and conrmed
further by microscopic observation as described by Munne-Bosch
and Alegre (2001). Chloroplasts-enriched fractions did not show
detectable activities of NADPH-cytochrome c reductase, latent
IDPase, vanadate-sensitive ATPase, and cytochrome c oxidase,
which are markers of the endoplasmic reticulum, Golgi apparatus,
plasma membrane, and mitochondrion, respectively.
For MDA analyses, leaf samples (500 mg) and chloroplasts
(50 mg) were extracted with 80:20 (v/v) ethanol/water using
ultrasonication (Vibra-Cell Ultrasonic Processor). After centrifug-
ing at 3,000 g for 10 min at 4 C, the pellet was re-extracted twice
with the same solvent. Supernatants were pooled and an aliquot of
appropriately diluted sample was added to a test tube with an equal
volume of either (i) TBA solution comprising 20% (w/v) trichlo-
roacetic acid and 0.01% (w/v) BHT, or (ii) +TBA solution
containing the above plus 0.65% (w/v) TBA. Samples were heated
at 95 C for 25 min and, after cooling, absorbance was read at
440 nm, 532 nm, and 600 nm. MDA equivalents (nmol ml1) were
calculated as 106((AB)/157,000), where A=((Abs 532+TBA)
(Abs 600+TBA)(Abs 532TBAAbs 600TBA)), and B=((Abs
440+TBAAbs 600+TBA)0.0571) (Hodges et al. 1999).
Photosynthesis
Photosynthesis was measured on leaves by determining chlorophyll
uorescence and leaf gas-exchange rates at 2-h intervals throughout
the day. The relative eciency of photosystem II photochemistry
(/PSII) was calculated from chlorophyll uorescence data, which
were obtained at natural incident PPFD with a portable mini-PAM
uorimeter (Walz, Eeltrich, Germany), by using the equations
described by Genty et al. (1989). Leaf gas-exchange rates were
measured at incident PPFD in the eld using a Licor 6200 (Li-Cor
Inc., Lincoln, Neb., USA). CO2 assimilation and stomatal con-
ductance rates were estimated from gas-exchange measurements
using the equations developed by von Caemmerer and Farquhar
(1981).
Pigment determination
Leaves were collected at predawn (1 h before sunrise), midday (at
maximum incident PPFD) and evening (1 h after sunset), imme-
diately frozen in liquid nitrogen and stored at 80 C until analysis.
The extraction and analysis of pigments was carried out as
610

described by Munne-Bosch and Alegre (2000). In short, leaves characterized by a hot, dry summer (Table 1). Plants
(500 mg) were ground in a mortar and extracted repeatedly with were subject to drought stress in July, 1-, 3- and 7-year-
85% (w/w) and 100% acetone using ultrasonication (Vibra-Cell
Ultrasonic Processor; Sonics & Materials Inc., Canbury, Conn., old plants showing similar reductions in RWC and leaf
USA). Pigments were analyzed by HPLC, using a Dupont non- hydration (H) of ca. 26% and 45%, respectively, com-
endcapped Zorbax ODS-5 lm column (250 mm long, 4.6 mm pared to May (Fig. 1).
diameter, 20% C; Teknokroma, St. Cugat, Spain), and acetonitrile/
methanol (85:15, v/v) and methanol/ethyl acetate (68:32, v/v) as
eluants A and B, respectively. The gradient used was: 014 min Table 1 Monthly precipitation, and maximum diurnal vapor
100% A, 1416 min decreasing to 0% A, 1628 min 0% A, pressure decit (VPD), maximum diurnal PPFD, and maximum
2830 min increasing to 100% A, and 3038 min 100% A. De- diurnal air temperature (Tair) during the measurement days from
tection was carried out at 445 nm (Spectralphotometer 430; Kon- January to July 2000. Cistus clusii plants grew under Mediterra-
tron, Zurich, Switzerland). Compounds were identied by their nean eld conditions and received water exclusively from rainfall
characteristic spectra and by co-elution with chlorophyll and car- throughout the study period. Before the experiment, plants received
otenoid standards, obtained from Fluka (Buchs, Switzerland) and 169.5 mm water during the preceding three months
Homan-La Roche (Basel, Switzerland), respectively.
Precipitation VPD PPFD Tair
(mm) (kPa) (lmol m2 s1) (C)
Determination of a-tocopherol
January 23.7 0.77 936 11.3
Leaves were collected at predawn (1 h before sunrise), midday (at March 32.5 1.04 1,393 16.7
maximum incident PPFD) and evening (1 h after sunset), imme- May 54.2 1.77 1,916 26.4
diately frozen in liquid nitrogen and stored at 80 C until analysis. July 0.7 1.70 1,852 31.3
The extraction and analysis of a-tocopherol were carried out as
described by Munne-Bosch et al. (1999), except that samples were
extracted with n-hexane instead of methanol. In short, leaves
(500 mg) were ground in a mortar and extracted repeatedly with
n-hexane containing 1 lg ml1 BHT using ultrasonication (Vibra-
Cell Ultrasonic Processor). a-Tocopherol was analyzed by HPLC
using an ODS Hypersil-5 lm column (Spectralphotometer 430;
Kontron) and acetonitrile/water (98:2, v/v) containing 0.2% of 2 M
citric acid as an eluant. a-Tocopherol was quantied through its
absorbance at 295 nm (Spectralphotometer 430; Kontron) and its
uorescence emission at 340 nm after excitation at 295 nm (Fluo-
rescence detector SFM 25; Kontron). a-Tocopherol (Sigma-Ald-
rich) was used for calibration.

Plant water status

Plant water status was determined by measuring the relative water

content (RWC) and hydration of leaves at predawn (1 h before
sunrise), midday (at maximum incident PPFD) and evening (1 h
after sunset). The relative leaf RWC was determined as 100
(FWDW)/(TWDW), and the hydration of leaves (H) was deter-
mined as (FWDW)/DW, where FW is the fresh matter, TW is the
turgid matter after re-hydrating the leaves for 24 h at 4 C in
darkness, and DW is the dry matter after oven-drying the leaves for
24 h at 80 C.

Statistical analyses

Statistical dierences between treatments, dierent days, or times

of day were analyzed by analysis of variance (ANOVA) using the
SPSS package (Chicago, Ill., USA). Dierences were considered
signicant when P<0.05 (probability level).

Results

We simultaneously analyzed lipid oxidation in leaves

and chloroplasts, the photosynthetic capacity of leaves,
the de-epoxidation state of the xanthophyll cycle (DPS),
chlorophyll content, and levels of chloroplastic antioxi-
dant defenses (i.e. b-carotene and a-tocopherol) in leaves
of the same ontogeny in 1-, 3- and 7-year-old Cistus
clusii Dunal plants growing in the eld from January to
July. The environmental conditions during the experi-
ment were typical of the Mediterranean climate,
Fig. 1 Changes in the relative water content (RWC), hydration (H)
and net photosynthesis (A), lipid oxidation (MDA levels) of leaves
of 1- (black circles), 3- (black squares) and 7-year-old (white circles)
Cistus clusii plants grown under Mediterranean eld conditions.
Data correspond to measurements made at midday. Each value is
the mean SE of six measurements
 611

Photosynthesis and lipid oxidation in leaves groups of plants, and MDA levels in 7-year-old plants
were ca. 2.8-fold higher than in 1- and 3-year-old plants
Net photosynthesis was reduced by ca. 25% in 7-year- throughout the study period.
old plants, whereas no dierences were observed
between 1- and 3-year-old plants throughout the study.
Dierences were observed irrespective of water status Antioxidant defenses and the xanthophyll cycle
and were only evident in the oldest plants (Fig. 1). Leaf
gas-exchange analysis showed that the age-dependent Levels of chlorophyll, b-carotene, a-tocopherol, the de-
reductions in net photosynthesis were caused not only by epoxidation state of the xanthophyll cycle (DPS) and
a higher stomatal closure, but also by reductions in the xanthophyll contents in 7-year-old plants were signi-
relative eciency of PSII photochemistry (/PSII) cantly (P<0.05, ANOVA) lower than in 1- and 3-year-
(Fig. 2). Reductions in net photosynthesis, caused either old plants throughout the study period (Fig. 4, Table 2).
by stomatal closure or by a lower /PSII, were also
observed in drought (July) in all age groups.
Mature non-senescing (7-year-old) plants manifested
enhanced lipid oxidation in leaves throughout the study,
as measured by MDA levels which were 2.7-fold higher
than those observed in 1- and 3-year-old plants (Fig. 1).
By contrast, no signicant dierences (P<0.05,
ANOVA) were observed between 1- and 3-year-old
plants, thereby indicating that enhanced lipid oxidation
is more evident as plants age. Lipid oxidation also in-
creased in drought in all the age groups.

Lipid oxidation in chloroplasts

The enhanced age-dependent lipid oxidation in leaves

was, at least partly, due to enhanced lipid oxidation
metabolism in chloroplasts. The amount of MDA in
chloroplasts increased as a consequence of aging, and as
a consequence of stress in July (Fig. 3). Chloroplasts
showed the highest lipid oxidation in July in the three
Fig. 3 Eect of plant age on the formation of MDA in leaf
chloroplasts of C. clusii plants grown under Mediterranean eld
conditions. Values from January (open bars) and July (closed bars)
in 1-, 3- and 7-year-old plants are compared. Data correspond to
measurements taken at midday. Each value is the mean SE of
three measurements

Fig. 2 Diurnal variations in net

CO2 assimilation rates (A),
stomatal conductance rates (Gs)
and relative quantum eciency
of PSII photochemistry (/PSII)
in the leaves of 1- (black sym-
bols) and 7-year-old (white
symbols) C. clusii plants grown
under Mediterranean eld con-
ditions during 2000. Statistical
dierences (P 0.05, ANOVA)
between 1- and 7-year-old
plants are indicated by an
asterisk. For clarity, values for
3-year-old plants, which were
not signicantly dierent from
those of 1-year-old plants
(P 0.05, ANOVA), are not
shown. Each value is the mean
SE of six measurements
612

The levels of chlorophylls in 7-year-old plants were ca.

Table 2 Eects of plant age on the ratio of chlorophyll a to chlorophyll b and the xanthophyll pigment composition of C. clusii leaves during January and July 2000. Values, given in
mg (g DW)1, correspond to measurements taken at midday. Chl a/b Ratio of chlorophyll a to chlorophyll b, L lutein, N neoxanthin, V violaxanthin, Z zeaxanthin, A antheraxanthin.
20% lower than in the other age groups. Aging reduced

0.01+0.00
0.010.00
0.010.00
both the area and dry weight of leaves, which resulted in
similar specic leaf weights in all age groups (data not
shown). Thus, the level of chlorophylls was reduced by

A
aging when expressed on both a dry-weight and an area
basis. The levels of b-carotene, a-tocopherol, and the

0.030.00
0.030.01
0.020.00
DPS in 7-year-old plants were also signicantly smaller
than in the other age groups throughout the study

Z
period, with reductions between 25 and 85%. The ratio
of chlorophyll a to chlorophyll b did not show age-as-

0.030.01
0.030.00
0.030.01
sociated changes, and lutein and neoxanthin changed in
parallel with chlorophylls and b-carotene, showing
similar age-dependent reductions (by ca. 25%)

V
throughout the study (Table 2). Thus, the pigment
content and chloroplastic antioxidant defenses of leaves

0.040.00
0.030.00
0.030.00
were reduced by plant aging, which was associated with
the higher lipid oxidation and lower photosynthetic rates

0.160.01
0.140.01
0.120.01
L

2.760.01
2.640.11
2.840.11
Chl a/b
July

0.020.00
0.020.00
0.010.00
A

0.050.00
0.050.01
0.020.00
Z

0.080.01
0.070.01
0.060.01
Data are means SE of four measurements taken at midday

0.070.00
0.080.01
0.060.00
N

0.360.01
0.330.01
0.260.01
L

2.750.04
2.850.05
2.670.04
January

Chl a/b

Fig. 4 Eect of plant age on chlorophyll a+b (Chl), b-carotene

(b-Car) and a-tocopherol (a-Toc) levels and de-epoxidation state of
the xanthophyll cycle [DPS=(Z+1/2A)/(V+Z+A)] in 1- (black
circles), 3- (black squares) and 7-year-old (white circles) C. clusii
plants grown under Mediterranean eld conditions from January
(years)

to July 2000. A Antheraxanthin, V violaxanthin, Z zeaxanthin.

Age

Each value is the mean SE of four measurements

1
3
7
 613

observed in 7-year-old plants. The lower /PSII and DPS

levels in 7-year-old plants indicate plant aging caused
transient photoinhibitory damage to the photosynthetic
apparatus at midday.
In July, b-carotene, lutein and neoxanthin also
decreased in parallel with chlorophylls, whereas
a-tocopherol followed a distinct pattern (Fig. 4,
Table 2). The highest levels of a-tocopherol were ob-
served in July, which was associated with drought.
However, high levels of a-tocopherol were also observed
in January, which was probably associated with the low
temperature (Fig. 4, Table 1). The reductions in /PSII in
drought in all age groups were associated with increases
in the DPS, which indicates that the reductions of
photosynthesis are associated with the photoprotection
conferred by the xanthophyll cycle in drought. The di-
urnal variations in the parameters analyzed occurred
irrespective of plant age (data not shown).

Lipid oxidation increases progressively

in mature non-senescing plants

Levels of MDA, b-carotene, a-tocopherol, and the DPS

were analyzed simultaneously on one clear, sunny day
per month over 18 months in 7-year-old (mature) plants
(Fig. 5). MDA levels increased progressively throughout
the study period. MDA levels increased during the
summer, but did not revert completely when the stress
was released. Instead, MDA accumulated in a slow,
progressive manner, indicating that oxidative stress is
cumulative as mature plants age. The antioxidant and
photoprotective defenses of plants showed a marked
seasonal variation, displaying the lowest levels of
b-carotene and the highest levels of both a-tocopherol Fig. 5 Seasonal variations in levels of malondialdehyde (MDA), b-
carotene (b-Car) and a-tocopherol (a-Toc), and the de-epoxidation
and DPS during the summer. However, b-carotene, state of the xanthophyll cycle (DPS) in 7-year-old C. clusii plants
a-tocopherol, and the DPS were smaller during the grown under Mediterranean eld conditions. Data correspond to
summer of 2000, compared to the summer of 1999, measurements made on one clear, sunny day per month at midday
showing that antioxidant defenses tend to decrease from April 1999 to August 2000. Each value is the mean SE of
four measurements
progressively as plants age.

oxidative stress in plants. Mature C. clusii plants did not

Discussion
Three stages of development can be dierentiated in
plants (i.e. juvenile, transition, and mature) depending
on the reproductive phase (Bond 2000). The age-
associated changes observed in this study are the con-
sequence of plant aging, which is a slow, progressive and
cumulative process that does not immediately lead to
plant death. In our study, 7-year-old plants were still in a
reproductive stage, the age-related changes observed
were cumulative, and did not immediately lead to death,
either in cells, leaves or the whole plants, indicating that
the changes were caused by plant aging before senes-
cence occurred. The slow, progressive increases observed
in MDA in mature plants (Fig. 5) are indicative of the
nature of the process studied here, thus supporting the
contention that aging is caused by the accumulation of
show any symptom of senescence during or after the
study period, showing a slow, and progressive accumu-
lation of oxidative stress. In animals, it has been shown
that age-associated changes are small early in life but
rapidly increase with age because of the exponential
nature of the process (Harman 1981). This is what
appeared to occur to C. clusii plants. Age-associated
changes were only observed in mature (7-year-old)
plants, whereas no signicant changes were observed in
young (3-year-old) plants (Figs. 1, 3, 4).
Many theories have been propounded to account for
aging in animals (Warner et al. 1987; Medvedev 1990).
Although there are other theories that cannot be
excluded, our results support the free-radical theory of
aging, which postulates that age-associated changes are
the result of the accumulation of alterations induced by
free radicals within the cell (Harman 1981, 1991). It is
614
thought that in animals these alterations occur in
mitochondria because these are the organelles subjected
to the highest production rates of free radicals (Ashok
and Ali 1999; Rustin et al. 2000). In plants, however,
chloroplasts are the organelles most exposed to oxygen
toxicity because they function at high oxygen and in the
light (Halliwell and Gutteridge 1989), thus we hypoth-
esized that chloroplasts play a role in plant age-induced
oxidative stress.
Lipid oxidation analyses indicated that oxidative
lipid catabolism in chloroplasts was higher in mature
plants than in the younger ones, thereby indicating that
age-associated alterations in plants are mirrored, at least
in part, at the chloroplast level. MDA levels in chloro-
plasts were, however, only 2-fold higher than in leaves
(Figs. 1, 3), indicating that chloroplasts were not the
only target of oxygen toxicity, and that oxidative stress
in other organelles may be responsible for aging in
plants. The age-dependent increase in oxidative stress in
chloroplasts was also mirrored by the smaller amounts
of pigments (chlorophylls and carotenoids) and chloro-
plastic antioxidant defenses (b-carotene, a-tocopherol)
found in mature plants than in the younger ones
(Fig. 4).
The increases in MDA levels in chloroplasts suggest
that the plant age-dependent chlorophyll loss observed
in this study was caused, directly or indirectly, by
enhanced oxidative stress in chloroplasts. The role of
free radicals in chlorophyll loss is still poorly under-
stood, although it has been proposed that chlorophylls
can be degraded enzymatically through a light- and
oxygen-dependent process called photooxidation (Wise
and Naylor 1987; Takamiya et al. 2000). a-Tocopherol,
which protects the lipid matrix of photosynthetic mem-
branes by scavenging singlet oxygen and lipid peroxy
radicals (Fryer 1992; Havaux et al. 2000), has been
identied as a potent antioxidant that slows down the
eects of stress and senescence in plants (Thompson et al.
1987; Munne-Bosch et al. 1999). Thus, smaller amounts
of a-tocopherol in 7-year-old plants not only indicate an
age-dependent increase in oxidative stress, but also an
age-dependent decrease in antioxidant defenses, which,
in turn, may shift the pro-oxidative/antioxidative
balance in favor of the former, and thus exponentially
increase oxidative stress and aging.
The age-dependent increase in oxidative stress in
chloroplasts was observed in parallel with a lower DPS
and a reduced photosynthetic activity. The formation of
zeaxanthin from violaxanthin is associated with the
dissipation of excess energy in the pigment bed of
thylakoids (Demmig-Adams and Adams 1992), depends
on the generation of a proton gradient and ascorbate
disposability in thylakoids (Eskling et al. 1997), and
might be inuenced by the aggregation of light-har-
vesting complexes (Horton et al. 1996). Mature plants
showed lower /PSII associated with a lower DPS at
midday, when compared to the younger ones (Figs. 2,
4). This indicates that age-dependent and transient
photoinhibitory damage to the photosynthetic appara-
tus occurred, and was most likely associated with the
enhanced oxidative stress observed in mature plants.
Oxidative stress in chloroplasts may result in a de-reg-
ulation of several processes (e.g. PSII photochemistry,
xanthophyll cycle, antioxidant defenses), which may
directly or indirectly inuence the photosynthetic activ-
ity of leaves (Asada 1999).
In summary, this study shows that (i) oxidative stress
is associated with the aging in plants before senescence
occurs, (ii) oxidative stress accumulates in a slow,
progressive manner as plants age, (iii) the contribution
of oxidative stress to aging increases as plants age, and
(iv) the oxidative stress associated with aging occurs,
probably among other plant cell organelles, in chloro-
plasts.
Acknowledgements This research was supported by the Ministerio
de Ciencia y Tecnolog a (MCYT BOS2000-0560). We are very
grateful to Homan-La Roche for kindly providing us with lutein,
zeaxanthin and b-carotene, and to the Serveis Cient co-Tecnics
and the Serveis dels Camps Experimentals (University of
Barcelona) for technical assistance.
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