Functional
Ecology 1997
11, 416–424
Light stress effects and antioxidative protection in two
desert plants
P. STREB,* E. TEL-OR† and J. FEIERABEND*
*Botanisches Institut der Universität Frankfurt, Postfach 11 19 32, 60054 Frankfurt, Germany and †The
Hebrew University of Jerusalem, Faculty of Agriculture, PO Box 12, Rehovot 76100, Israel
Summary
1. The enzyme catalase was investigated as a sensitive marker of light stress in Retama
raetam stems and Atriplex halimus leaves. While the activity of catalase was readily
photoinactivated in vitro when crude extracts from these tissues were exposed to a photon flux of 500 µmol m–2 s–1 photosynthetic active radiation (PAR), no apparent losses
of the extractable catalase activities were observed when tissues were harvested from
desert plants at noon when light levels were as high as 2200 µmol m–2 s–1 PAR.
2. When stems of R. raetam or leaves of A. halimus were exposed to natural daylight
of 1000 µmol m–2 s–1 PAR in the presence of cycloheximide (CHI), in order to prevent
the new synthesis of catalase, or at a temperature of 42–44 °C, only minor losses of
catalase activity occurred in stems of R. raetam, but a marked apparent photoinactivation of catalase was observed in leaves of A. halimus.
3. Similarly, stronger declines of the ratio Fv/Fm, indicating more severe photoinhibition of photosystem II (PSII), were observed in A. halimus, compared with R. raetam.
4. For R. raetam, activities of some antioxidative enzymes and the carotenoid contents were higher in plants growing in the desert than in plants collected from nondesert locations.
5. During the daily increase of light and temperature the antioxidants ascorbate and
glutathione became more oxidized and violaxanthin was converted to zeaxanthin in R.
raetam stems, while these antioxidants became more reduced and zeaxanthin was not
notably accumulated in A. halimus leaves.
6. The results suggest that the desert plants R. raetam and A. halimus apply different
strategies of protection against light-stress damage. In the stems of R. raetam antioxidative protection appears to play the major role, while A. halimus appears to avoid
light damage in the field by the mutual shading of leaves and reflection of light at the
leaf surface.
Key-words: Adaptation to stress, antioxidants, carotenoids, catalase, photoinactivation
Functional Ecology (1997) 11, 416–424
Introduction
© 1997 British
Ecological Society
Plants growing in desert areas, such as Retama raetam
(Forsk.) Webb. and Atriplex halimus (L.) that occur in
the Negev (Israel), have to survive at extremely high
irradiance, air temperature and severe soil drought.
Because of stomatal closure under extreme drought
conditions, overreduction of the photosynthetic electron transport chain will increase the generation of
reactive oxygen species and give rise to oxidative
damage (Mishra & Singhal 1992; Quartacci & NavariIzzo 1992; Moran et al. 1994; Foyer, Lelandais &
Kunert 1994; Sgherri & Navari-Izzo 1995). Therefore,
desert plants must be specifically adapted to the environment, either by anatomical characteristics that
enable them to lower the intercept of solar radiation, or
by biochemical means which ensure that photodamage
is either avoided or rapidly repaired.
The shrubs of R. raetam produce new leaves only
during the more favourable spring season and are
deciduous during the summer drought period (Stocker
1974b). Atriplex halimus is a highly salt-tolerant plant
which secretes salt into the vacuoles of hair bladder
cells. When these cells collapse they leave a strongly
light-reflective and non-wettable layer of salt crystals
and wax on the leaf surface (Osmond, Björkman &
Anderson 1980; Freitas & Breckle 1992).
Furthermore, vertical leaves of A. halimus intercept
considerably less of the solar radiation, as has been
shown for twig desert shrubs (Ehleringer & Cooper
1992) and the leaves are mostly shaded by other
leaves within the shrub (Osmond et al. 1980).
416
417
Light stress and
adaptation
© 1997 British
Ecological Society,
Functional Ecology,
11, 416–424
Tolerance to photo-oxidative stress may also be
acquired by various strategies of physiological adaptation. While carotenoids are, in general, efficient
quenchers of the triplet excitation state of chlorophyll
and of singlet oxygen (Asada & Takahashi 1987), the
xanthophyll zeaxanthin in particular, facilitates the
harmless dissipation of light energy as heat (DemmigAdams & Adams 1992). Besides, both the chloroplast
and cytoplasmic compartment of leaves contain cascades of enzymes and antioxidative metabolites which
allow the efficient detoxification of reactive oxygen
species. Superoxide, which is generated in the Mehler
reaction, is dismutated to H2O2 by superoxide dismutase (SOD). The resulting H2O2 is reduced by ascorbate peroxidase with the production of monodehydroascorbate. Reduced ascorbate is regenerated by the
reduction of monodehydroascorbate with ferredoxin
or NADPH or by the reduction of dehydroascorbate
with reduced glutathione. Finally, glutathione can be
reduced by glutathione reductase with NADPH as
electron donator. Additional peroxidases with different substrate specificity may also consume H2O2.
Furthermore, ascorbate is involved in the deep-oxidation of violaxanthin to zeaxanthin and the regeneration of the radical scavenger α-tocopherol and can
react non-enzymatically with most reactive oxygen
species (Asada & Takahashi 1987; Foyer et al. 1994).
During photorespiration, which is also important to
prevent an overreduction of the photosynthetic electron transport chain (Heber et al. 1996), H2O2 is produced in the peroxisomes by glycolate oxidase and
removed by the enzyme catalase (Asada & Takahashi
1987).
In spite of the multitude of protective mechanisms,
the peroxisomal enzyme catalase and the PSII reaction centre appear to exhibit a preferential light-sensitivity in leaves of most plants investigated so far
(Feierabend, Schaan & Hertwig 1992; Aro, Virgin &
Andersson 1993). The enzyme catalase is continuously inactivated by visible light in leaves but under
non-stressed conditions the loss by photoinactivation
can be compensated for by new synthesis. Additional
stress factors, such as heat, cold, salt or toxic chemicals, may, however, either enhance the rate of catalase
inactivation or inhibit protein synthesis, with the
result that the loss of catalase activity exceeds the
capacity for its new synthesis. Therefore, apparent
photoinactivation of catalase represents an early
widespread stress symptom in light, which usually
accompanies photoinhibition of PSII and precedes the
appearance of more general oxidative damage (Volk
& Feierabend 1989; Feierabend et al. 1992; Hertwig,
Streb & Feierabend 1992; Streb, Michael-Knauf &
Feierabend 1993; Streb & Feierabend 1996).
Consequently non-adapted plants can be expected to
suffer from severe declines of both catalase and PSII
activity under desert conditions.
We have, therefore, now investigated whether photoinactivation of catalase, as indicator of early photo-
damage, and photo-oxidative chlorophyll bleaching
occurred in the desert plants R. raetam and A. halimus
during the dry season when extremely high temperature and sunlight irradiance were prevailing. As
potential components involved in oxidative stress protection and tolerance mechanisms, the contents of
ascorbate and glutathione, the deep-oxidation of the
carotenoid violaxanthin to zeaxanthin and the activities of several antioxidative enzymes were assayed.
Previous investigations had already indicated, that
high amounts of antioxidants and high activities of
antioxidative enzymes were accumulated in R. raetam
during the spring season (Mittler, Nir & Tel-Or 1991).
Materials and methods
PLANT MATERIAL AND SITE SELECTION
Stems of R. raetam and leaves of A. halimus were collected at different times of day in Israel between
October and December 1992. Both species were analysed at Sede Boqer and three other sites of the Negev
desert near Sede Boqer. In addition, stems of R. raetam were collected within the campus of the Faculty
of Agriculture in Rehovot, and leaves of A. halimus
were collected at Palmahim near the Mediterranean
sea (c. 20 km from Rehovot). At both locations clouds
were frequently observed. Variations of light intensities during the period of the experiments were in the
range of 500 to 1500 µmol m–2 s–1 PAR and maximum
variation of air temperature was limited to a range of
25 to 35 °C between 09.00 h and 17.00 h. In Sede
Boqer no clouds were observed during the investigation period and climatic conditions were constant. The
light intensity increased from 750 µmol m–2 s–1 PAR
in the morning (07.00–08.00 h) to 2200 µmol m–2 s–1
PAR at noon (12.00–13.00 h) and then decreased to
10 µmol m–2 s–1 PAR in the evening (17.00–18.00 h).
The air temperature was 25 °C in the morning, 45 °C
at noon and 32 °C in the evening. At all locations no
rainfall occurred during the period of our investigations. Mean environmental conditions and precipitation of the Negev were described by Mittler et al.
(1991).
Plant material collected from the three desert locations was kept on ice in darkness for 4 h before analysis. Plant material collected at Palmahim was transported to the laboratory on wet filter paper and
analysed. Plant material from Rehovot and Sede
Boqer was analysed immediately after harvest.
EXPERIMENTAL TREATMENTS
Stems of R. raetam and leaves of A. halimus were
excised, carefully washed and placed in Petri dishes in
water in the absence (control) or presence of 35·5 µM
cycloheximide (CHI), to block the new synthesis of
catalase, or of 6·3 mM chloramphenicol (CAP), to
block the new synthesis of chloroplastic proteins.
418
P. Streb et al.
Retama raetam stems were kept in darkness for 10 h
(for enzyme and chlorophyll determinations) or 1 h
(for fluorescence measurements), A. halimus leaves
were kept in darkenss 14 h before light treatment.
Stems and leaves were exposed to natural sunlight of
an average photon flux of 1000 µmol m–2 s–1 PAR at
25–30 °C, or at 42–44 °C to mimic high temperature
stress, in open Petri dishes with water in the presence
or absence of inhibitors. The durations of the light
treatments were 3 h for assays of chlorophyll fluorescence and 7 h for enzyme activity measurements and
chlorophyll determination. In addition, stems of R.
raetam were incubated in the presence of 177 µM CHI
and in the presence or absence of CAP at
1300 µmol m–2 s–1 PAR (Phillips mercury lamp) at
28 °C. For the in vitro assay of catalase photoinactivation, supernatants from crude extracts were exposed to
520 µmol m–2 s–1 PAR (Phillips mercury lamp) at
28 °C in small test tubes.
FLUORESCENCE MEASUREMENTS
The ratio of variable to maximum fluorescence
(Fv/Fm) of stems or leaves was determined with a
PAM 101 fluorometer (Walz, Germany) after 10 min
dark adaptation. Fo was determined after irradiating
tissues with modulated light of 0·1 µmol m–2 s–1 PAR.
Fm was determined after excitation with saturating
light of 2400 µmol m–2 s–1 PAR (Kaltlicht KL
1500 T). The ratio Fv/Fm was calculated by a personal
computer with DA version 2 software. The decline in
the ratio Fv/Fm was regarded as a measure of photoinhibition of PSII (Krause & Weis 1991).
ANALYTICAL METHODS
© 1997 British
Ecological Society,
Functional Ecology,
11, 416–424
Stems and leaves (c. 0·4 g fresh weight) were ground
with mortar and pestle in 2 ml of 50 mM potassiumphosphate buffer, pH 7·5, for the assay of enzyme
activities, or in 1% (w/v) metaphosphoric acid for the
determination of antioxidants. The extract volume
was adjusted to 5 ml and centrifuged at 4 °C for 5 min
at 25 000 g for the enzyme measurements and either
for 25 min at 38 000 g or for 30 min at 25 000 g for the
antioxidant determinations. Clear supernatants were
used for all assays.
The assays of catalase, glutathione reductase, guaiacol peroxidase and glycolate oxidase activity and
the determination of the chlorophyll content were carried out as already described by Streb et al. (1993).
Ascorbate peroxidase and superoxide dismutase were
assayed as described by Mittler et al. (1991). The protein content was measured by the method of Bradford
(1976). The determination of reduced and oxidized
ascorbate and glutathione was carried out as described
by Streb & Feierabend (1996).
For pigment extraction c. 0·4 g fresh weight of plant
material was frozen in liquid nitrogen, stored at
–20 °C and extracted in 5 ml dimethylformamide
under an atmosphere of nitrogen at 4 °C for 5 days as
described by Bergweiler & Lütz (1986). After filtration through 0·45-µm filters of Nalgene pigments were
separated by HPLC according to Thayer & Björkman
(1990), applying a C18 pre-column (Machery &
Nagel) and a C18 non-endcapped reversed phase column (Waters Resolve, 3·9 × 300 mm). Pigments were
eluted with a solvent gradient progressing from 100%
acetonitril/methanol (85/15, v/v) to 100% methanol/
ethylacetate (68/32, v/v), at flow rates of 0·55 to
1 ml min–1. For identification the retention times were
compared with authentic zeaxanthin (Roth), lutein
(Roth) and β-carotene (Merck) standards. Other reference pigments were prepared by thin-layer chromatography as described by Hager & MeyerBertenrath (1966) from spinach leaves, identified by
absorption spectra and quantified by their specific
extinction coefficients (Davies 1976). From the peak
areas obtained after HPLC separation of known
amounts of pure standards pigments conversion factors were determined for the quantitative estimation of
pigment contents.
Results are expressed as mean values of at least
three independent experiments. Standard errors of the
mean are indicated. For statistical analysis the
Student’s t-test was applied.
Results
PHOTOINACTIVATION OF CATALASE AND PSII
High light intensities, high air temperatures and soil
drought are the prevailing environmental conditions
in the Negev desert in early October. Such conditions
would be expected to induce stomatal closure and,
owing to the resulting CO2-deficiency, severe oxidative stress in non-adapted plants. Therefore, we examined the occurrence of photoinactivation of catalase,
which represents an early stress symptom in the light,
and the destruction of chlorophyll in stems of R. raetam and leaves of A. halimus in the desert. During the
daily increase in light intensity no loss of catalase
activity was observed but the activity of this enzyme
even increased markedly in the stems of R. raetam
(Fig. 1a). The chlorophyll content slightly increased
during the day in both species (Fig. 1b). In order to
examine the photoinactivation of catalase from these
plants we exposed supernatants of crude extracts from
stems and leaves to light. In cell-free extracts from
both plant species catalase was readily inactivated
during a 5-h incubation in light but not in the dark
(Fig. 2). This indicates that the properties of the catalases from these desert plants closely resembled those
known from other species.
In order to discriminate between various strategies
of light protection, detached stem segments and
leaves were exposed to direct sunlight in the presence
and absence of the protein synthesis inhibitor CHI. In
the absence of CHI no significant photoinactivation of
419
Light stress and
adaptation
catalase was observed in stems of R. raetam and only
a minor decrease was observed in leaves of A. halimus
(Fig. 3a). In the presence of 35·5 µM CHI catalase
activity declined slightly in stems of R. raetam, but
the decline was stronger in leaves of A. halimus
accounting for a loss of about 26% (Fig. 3a). Similar
observations were made when R. raetam stems were
incubated in the presence of 177 µM CHI in order to
confirm that the inhibitor concentrations were high
enough (Table 1). In darkness catalase activity did not
Fig. 1. Changes of (a) catalase activity and (b) chlorophyll content in stems of Retama
raetam (l) and leaves of Atriplex halimus (●) during the course of the day in the Negev
desert at Sede Boqer. Plant material was collected at 07.00 to 08.00 h (morning), 12.00
to 13.00 h (noon) or at 17.30 to 18.30 h (evening) and analysed immediately.
Fig. 2. Decline of catalase activity as a percentage of initial activity during illumination of crude soluble extracts of (a) Retama raetam stems and (b) Atriplex halimus
leaves with light of 520 µmol m–2 s–1 PAR (l ). Control extracts were kept in darkness (●) at 28 °C.
© 1997 British
Ecological Society,
Functional Ecology,
11, 416–424
decline in stems of R. raetam and leaves of A. halimus
during prolonged incubations of 10–14 h in the presence of CHI (Table 1). During incubation at an elevated temperature of 42–44 °C in light, catalase was
strongly inactivated, losing 46% of its activity in
leaves of A. halimus (P < 0·01), while only a considerably smaller decline was observed in stems of R. raetam (Fig. 3a). As an alternative peroxide scavenging
and heme containing enzyme guaiacol peroxidase were
also investigated. The activity of guaiacol peroxidase
did not decrease under the different incubation conditions, but increased in the control samples and during
incubation at 42–44 °C in leaves of A. halimus. The
latter observation suggests that a temperature of
42–44 °C did not yet act as heat-shock blocking
enzyme synthesis (Fig. 3b). The chlorophyll content
declined in leaves of A. halimus during exposures to
CHI or to high temperature by 30%, while the chlorophyll content of R. raetam stems was hardly affected
(Fig. 3c).
The occurrence of photoinhibition of PSII was
investigated, for comparison, by chlorophyll fluorescence measurements. Within 3 h PSII activity
declined by 28% in excised stems of R. raetam in
the absence of inhibitor at 25–30 °C. In the presence
of CAP and at elevated temperature the Fv/Fm ratio
decreased significantly in stems of R. raetam by
56% (P < 0·05) or 40% (P < 0·10), respectively (Fig.
4a). In leaves of A. halimus photoinactivation of
PSII was stronger under all conditions, relative to R.
raetam. In controls without inhibitor and at 42 °C
PSII activity declined by 50%. At high temperature
the decline was significantly higher, as compared
with R. raetam (P < 0·10). In the presence of CAP
the ratio Fv/Fm declined by 70% within 3 h (Fig.
4b). A set of later experiments confirmed the low
susceptibility of R. raetam stems to photoinactivation of PSII, both under controlled artificial light
and in natural sunlight. In these experiments the
ratio Fv/Fm did not decline in the control treatments
and only minor declines were observed in the presence of CAP (Table 2).
Fig. 3. Change of (a) catalase and (b) guaiacol peroxidase activity and of (c) chlorophyll content as a percentage of initial value
during 7-h exposure of stem segments of Retama raetam and leaves of Atriplex halimus in the presence or absence of 35·5 µM
cycloheximide at 25–30 °C or at 42–44 °C in natural sunlight of approximately 1000 µmol m–2 s–1 PAR.
420
P. Streb et al.
Table 1. Changes of catalase activity (µkat g–1 fresh weight) during 10 h (Retama raetam) or 14 h (Atriplex halimus) dark
incubation in the presence or absence of CHI and subsequent 7 h exposure to sunlight
Retama raetam
Before treatment
10–14 h dark
7 h sunlight
Atriplex halimus
Control
35·5 µM CHI
177 µM CHI
Control
35·5 µM CHI
14·0 ± 2·0
19·1 ± 2·6
19·0 ± 2·7
14·0 ± 2·0
16·0 ± 2·1
13·3 ± 1·7
14·0 ± 2·0
16·5 ± 2·9
14·3 ± 1·7
8·9 ± 1·2
11·2 ± 0·8
9·5 ± 1·5
8·9 ± 1·2
9·6 ± 0·9
7·1 ± 1·1
Fig. 4. Changes of the ratios of variable to maximum chlorophyll fluorescence Fv/Fm
as a percentage of initial measurement in (a) stems of Retama raetam and (b) leaves
of Atriplex halimus during incubation in natural sunlight of approximately
1000 µmol m–2 s–1 PAR in the presence (n) or absence (l) of 6·3 mM chlorampenicol
at 25–30 °C, or at a temperature of 42–44 °C (s). Initial ratios of Fv/Fm after dark
incubation were 0·75 ± 0·01 for stems of R. raetam in the presence and absence of
CAP and 0·73 ± 0·04 and 0·72 ± 0·04 for leaves of A. halimus in the presence and
absence of CAP respectively.
Table 2. Changes of the ratio of variable to maximum chlorophyll fluorescence
Fv/Fm as a percentage of initial measurements in stems of Retama raetam during
incubation under controlled conditions with a constant irradiance of
1300 µmol m–2 s–1 PAR at 28 °C (n = 4) and in natural sunlight of approximately
1000 µmol m–2 s–1 PAR at 25–30 °C (n = 2). Experiments were performed 1 month
later than the experiments shown in Fig. 4. Initial ratio of Fv/Fm was 0·79 ± 0·02 after
the dark pretreatment in water or CAP
1h
3h
Controlled light conditions
Natural sunlight
Control
CAP
Control
CAP
96·2 ± 2·5
94·9 ± 1·3
84·8 ± 2·5
83·5 ± 2·5
89·9 ± 1·3
94·9 ± 1·3
89·9 ± 2·5
89·9 ± 2·5
tents were essentially similar in plants of both species
grown under desert or non-desert conditions. In R.
raetam stems the activities of catalase and ascorbate
peroxidase did not differ greatly between stems collected in Rehovot or at sites near Sede Boqer, but
catalase activity showed some variation between different collection sites in the desert. The activities of
SOD and guiacol peroxidase were much higher in
stems collected in Sede Boqer, as compared with stems
collected in Rehovot, while activities of glutathione
reductase and glycolate oxidase were lower in plants
from the desert locations. In stems of R. raetam the
content of ascorbic acid and the ratio of reduced to
oxidized ascorbic acid was remarkably higher at the
desert site, as compared with the Rehovot site, whereas
the concentration of glutathione was similar. The concentration of carotenoids and, in particular, that of
xanthophyll-cycle pigments was much higher in stems
collected at the desert site, as compared with the
Rehovot site.
In leaves of A. halimus plants collected at the desert
site, activities of SOD, ascorbate peroxidase and guiacol peroxidase were up to threefold higher, relative to
leaves collected at the Palmahim site, but no major
differences were observed for other enzyme activities.
The contents of ascorbate, glutathione, carotenoids
and xanthophyll-cycle pigments were essentially similar for all collection sites.
On the basis of fresh weight or of the chlorophyll
content the activities of most antioxidative enzymes
and the contents of ascorbate and total and xanthophyll-cycle carotenoids were higher in desert-grown
plants of R. raetam than of A. halimus (Table 3).
COMPARISON OF ANTIOXIDATIVE ENZYMES,
© 1997 British
Ecological Society,
Functional Ecology,
11, 416–424
ANTIOXIDANTS AND CAROTENOIDS OF PLANTS
DAILY FLUCTUATIONS OF CAROTENOIDS AND
FROM DIFFERENT SITES
ANTIOXIDANTS
Plants growing in the desert have to cope with a
higher probability of oxidative damage, as compared
with plants growing under more favourable environmental conditions. In order to investigate whether systems of antioxidative protection were enhanced under
extreme desert conditions, we compared plants growing at a desert site near Sede Boqer to plants growing
at Rehovot and Palmahim (Table 3).
Structurally, desert plants were smaller than plants
growing at a non-desert location. However, on a
fresh-weight basis the protein and chlorophyll con-
In order to further characterize the response of the
plants to the daily changes of light and temperature in
the desert, fluctuations of xanthophyll-cycle pigments
(VAZ) and their deep-oxidation state, which may
reflect the adaptation to high light intensity, and of the
oxidation of ascorbate and glutathione were investigated during the course of the day. In stems of R. raetam the VAZ content declined markedly during the
day, whereas only a slight reduction was measured in
leaves of A. halimus. At noon the amount of zeaxanthin increased up to 70% of the VAZ content in stems
421
Light stress and
adaptation
Table 3. Comparison of enzyme activities of peroxide metabolism and of the contents of protein, antioxidants and pigments in
stems of Retama raetam and leaves of Atriplex halimus, collected at similar times of day at different sites in Israel. Data are
expressed on a fresh weight (FW) basis. Plants were collected in the Negev desert near Sede Boqer, in Rehovot (R. raetam) or
at Palmahim (A. halimus). Glycolate oxidase (G. oxidase); guaicol peroxidase (G. peroxidase); ascorbate peroxidase (A. peroxidase); glutathione reductase (G. reductase); superoxide dismutase (SOD); ratio of reduced to oxidized ascorbic acid
(AA/DHAA) or of reduced to oxidized glutathione (GSH/GSSG); Violaxanthin + Antheraxanthin + Zeaxanthin (VAZ); ND
(not determined). Each value represents the mean of at least three different experiments
Retama raetam
Rehovot
Protein (mg g–1 FW)
16·0 ± 0·8
Catalase (µkat g–1 FW)
14·0 ± 2·0
G. oxidase (nkat g–1 FW)
G. peroxidase (nkat g–1 FW)
10·2 ± 0·8
445·9 ± 44·6
SOD (units g–1 FW)
A. peroxidase (nkat g–1 FW)
441·6 ± 110·8
358·9 ± 52·4
G. reductase (nkat g–1 FW)
Ascorbate (µmol g–1 FW)
AA/DHAA
Glutathione (nmol g–1 FW)
GSH/GSSG
Chlorophyll (µg g–1 FW)
13·2 ± 0·8
3·1 ± 0·3
0·44 ± 0·14
311·6 ± 37·6
6·93 ± 3·01
676·1 ± 23·8
Carotenoids (nmol g–1 FW)
VAZ/Chl (mmol/mol)
VAZ/Carotenoids%
174·1
33·6 ± 5·6
13·0
Atriplex halimus
Sede Boqer
14·7 ± 1·7
(13·8–21·9)*
13·2 ± 2·0
(9·7–25·2)*
3·4 ± 1·3
770·8 ± 95·6
·(358–612)*
1158·0 ± 126·0
ND
(239–401)*
5·5 ± 0·7
6·1 ± 1·5
1·30 ± 0·58
365·7 ± 34·6
9·87 ± 4·08
401·1 ± 25·8
(422–687)*
241·1
166·2 ± 26·4
25·6
Palmahim
4·0 ± 1·2
8·9 ± 1·2
3·4 ± 0·7
4·2 ± 1·1
119·2 ± 28·6
101·4 ± 29·2
16·2 ± 2·1
3·6 ± 0·3
9·04 ± 3·46
406·9 ± 53·4
3·01 ± 0·32
442·5 ± 85·9
143·1
51·0 ± 15·3
10·0
Sede Boqer
4·9 ± 0·8
(4·8–6·9)*
6·8 ± 1·2
(4·8–6·0)*
4·1 ± 1·3
18·4 ± 2·3
(8·7–10·0)*
262·8 ± 27·3
ND
(206)*
14·0 ± 7·4
4·3 ± 0·6
2·64 ± 0·17
397·1 ± 20·2
3·09 ± 0·62
451·2 ± 9·4
(302–557)*
141·8
62·0 ± 9·5
10·6
* Data in brackets indicate additional measurements with plants from different collection sites in the Negev desert and illustrate the range of variation observed.
of R. raetam, while little change occurred in leaves of
A. halimus (Fig. 5a,b). The total contents of glutathione and ascorbate and, in particular, of their
reduced forms significantly declined in stems of R.
raetam during the course of the day (Fig. 5c–f). At
noon a strong increase of oxidized glutathione accompanied the decline in reduced glutathione in stems of
R. raetam (Fig. 5d,f). In leaves from intact plants of A.
halimus the contents of reduced ascorbate and glutathione increased during the day (Fig. 5c,d).
Discussion
© 1997 British
Ecological Society,
Functional Ecology,
11, 416–424
High light intensities, high air temperatures and
extreme drought are known to induce photoinactivation of both catalase and PSII in various plant
species (Ludlow 1987; Feierabend et al. 1992;
Cornic 1994). However, in the photosynthesizing
stems of R. raetam and leaves of A. halimus of intact
plants growing under the extreme natural desert
conditions, catalase activity did not decline, but
even increased in the stems of R. raetam (Fig. 1a).
Because in cell-free extracts of the two plants catalase activity was in vitro similarly susceptible to
photoinactivation (Fig. 2), as catalases from other
sources (Björn 1969; Cheng, Kellogg & Packer 1981;
Feierabend & Engel 1986), these desert plants must
be endowed with specific mechanisms of adapta-
tion, in order to avoid or prevent apparent losses of
catalase activity under natural conditions. Attempts
were made to elucidate mechanisms that contribute to
the extraordinary in vivo stability of catalase in the
two desert species. In both species excess irradiance is
to some extent avoided by plant morphology and
structure, as is illustrated by the loss of leaves in R.
raetam and by the vertical orientation and highly
reflecting surface of A. halimus leaves (Stocker
1974b; Osmond et al. 1980). However, after careful
removal of surface substances catalase activity was
still stable in stems of R. raetam and only a minor
apparent loss was induced in A. halimus leaves during
subsequent light exposure. When potential repair of
catalase degradation through new synthesis was
blocked by the application of the protein synthesis
inhibitor CHI, photoinactivation became apparent,
particularly in A. halimus leaves. Relative to results
obtained for several non-desert plants in the presence
of translation inhibitors (Feierabend & Engel 1986),
the rate of catalase photoinactivation was low, both in
R. raetam and A. halimus, suggesting that it was efficiently avoided in vivo. However, when the light
exposure was performed at high temperature a considerably larger decline of the catalase activity than in the
presence of CHI was induced in A. halimus, but not in
R. raetam. Thus under heat stress A. halimus appeared
to be more susceptible to light than R. raetam.
422
P. Streb et al.
Usually, high temperature is known to block protein
synthesis, except for specific heat-shock proteins
(Vierling 1991). Therefore, photoinactivation of
catalase (Feierabend et al. 1992) and photoinhibition of PSII (Ludlow 1987) were found to occur
under heat-shock conditions. A higher susceptibility
of A. halimus leaves to photoinactivation, as compared with R. raetam stems, was also confirmed by
measurements of chlorophyll fluorescence, indicating more severe photoinhibition of PSII both in the
presence and absence of the translation inhibitor
CAP and, in particular, at high temperature. Because
leaves of A. halimus were much less light sensitive at
the desert site, as compared with surface-washed,
wet leaves, the light-reflecting surface layer may
provide considerable protection under natural desert
conditions.
As much as 40–60% of the sunlight may be
reflected at the surface of the leaves of different
Atriplex species (Sinclair & Thomas 1970; Mooney,
Ehleringer & Björkman 1977). A similarly high light
reflectance of their leaf surfaces was considered to be
common among all desert species of Atriplex
(Osmond et al. 1980). When dry leaves of Atriplex
were rehydrated the light reflection decreased
Fig. 5. Changes of the contents of (a) the xanthophyll-cycle carotenoids violaxanthin + antheraxanthin + zeaxanthin (VAZ), (b) the relative zeaxanthin content (% of
VAZ), and of the reduced forms of (c) ascorbate (AA) and (d) glutathione (GSH) and
oxidized forms of (e) ascorbate (DHAA) and (f) glutathione (GSSG) in stems of
Retama raetam (l) and leaves of Atriplex halimus (●) during the course of a day.
strongly (Mooney et al. 1977). To the authors’ knowledge reflectance data are not available for R. raetam
stems. However, the anatomical structure of the R.
raetam stems, which was described by Stocker
(1974a), does not show any particular elements that
could enhance light reflection.
Mechanisms reducing light absorption are
reported to protect the photosystems from overreduction and photoinhibition under drought stress
(Cornic 1994). Robinson, Lovelock & Osmond
(1993) have shown that the removal of a lightreflecting wax coating increased the susceptibility to
photoinhibition in Cotyledon orbiculare. Lang &
Schindler (1994) have demonstrated that leaf hairs of
Senecio medley decreased high light-induced photoinhibition as well as the deep-oxidation of violaxanthin. Furthermore, the vertical orientation of
leaves protected twig desert shrubs from a loss of
photosynthetic quantum efficiency under excessive
light (Ehleringer & Cooper 1992). In R. raetam
anatomical adaptation and repair of damaged catalase cannot be the only means contributing to the
maintenance of high constant catalase activity in the
desert. These also appear to involve a high capacity
for antioxidative protection. High activities of
antioxidative enzymes, such as SOD and glutathione
reductase, and high levels of the antioxidants ascorbate and glutathione were induced under stress conditions and have been correlated with tolerance to
drought and light stress (Foyer et al. 1994).
Accordingly, in both R. raetam and A. halimus
activities of antioxidative enzymes and the contents
of antioxidants were higher in plants growing in the
desert, as compared with plants from a non-desert
location. High contents of zeaxanthin are assumed
to indicate a high capacity for the dissipation of
excess light energy as heat (Demmig-Adams &
Adams 1992). Overall, the capacity to dissipate
light energy by xanthophyll-cycle pigments and to
scavenge reactive oxygen species by carotenoids
and antioxidants were much higher in R. raetam
stems, as compared with A. halimus leaves on the
basis of fresh weight and chlorophyll content.
During the daily maximum of light and temperature
at the desert location a decrease in VAZ content, a
strong increase in the amount of zeaxanthin and an
oxidation of ascorbic acid and glutathione, which
indicates photo-oxidative stress (Foyer et al. 1994;
Streb & Feierabend 1996), were observed in R. raetam stems, while these parameters exhibited only
minor changes in A. halimus leaves. These findings
support the conclusion that the light-reflecting surface layer of the A. halimus leaves largely protected
this plant from light stress in the desert. While any
physical adaptation mechanism weakening the
absorbency of light will inevitably also reduce the
yearly carbon gain of photosynthesis (Ehleringer &
Cooper 1992), this is not applicable for R. raetam
stems. As was shown by Stocker (1974b), R. raetam
423
Light stress and
adaptation
stems are able to perform carbon assimilation for all
12 months of a year. Its capacity for anti-oxidative
protection is, however, highest during the growing
period in spring (Mittler et al. 1991). Therefore, it
remains to be investigated, whether high antioxidative defence capacities are associated with the ability to perform net carbon assimilation under extreme
stress conditions. Mechanisms of biochemical adaptation may allow plants to respond with higher flexibility to changing environmental conditions, than
adaptive strategies by merely anatomical characteristics would allow.
Acknowledgements
We thank Professor Dr B. Rubin for the use of his
PAM-fluorometer and Dr S. Boussiba and Dr A.
Vonshak for use of the laboratory facilities in Sede
Boqer. The help of Dr A. Rozen and S. Lechno during
the sampling of plant material and performing the
experiments is gratefully appreciated. Peter Streb
thanks the DAAD, Bonn, for financial support.
References
© 1997 British
Ecological Society,
Functional Ecology,
11, 416–424
Aro, E.M., Virgin, I. & Andersson, B. (1993)
Photoinhibition of photosystem II, inactivation, protein
damage and turnover. Biochimica et Biophysica acta
1143, 113–134.
Asada, K. & Takahashi, M. (1987) Production and scavenging of active oxygen in photosynthesis. Photoinhibition
(eds D. J. Kyle, C. B. Osmond & C. J. Arntzen), pp.
227–287. Elsevier Science Publishers, Amsterdam.
Bergweiler, P. & Lütz, C. (1986) Determination of leaf pigments by HPLC after extraction with N,NDimethylformamide: ecophysiological applications.
Environmental and Experimental Botany 26, 207–210.
Björn, L.O. (1969) Photoinactivation of catalases from
mammal liver, plant leaves and bacteria. Comparison of
inactivation cross sections and quantum yields at
406 nm.
Photochemistry
and
Photobiology 10,
125–129.
Bradford, M.M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Analytical
Biochemistry 72, 248–254.
Cheng, L., Kellogg, E.W. & Packer, L. (1981)
Photoinactivation of catalase. Photochemistry and
Photobiology 34, 125–129.
Cornic, G. (1994) Drought stress and high light effects on
leaf photosynthesis. Photoinhibition of Photosynthesis
from Molecular Mechanisms to the Field (eds N. R. Baker
& J. R. Bowyer), pp. 297–314. Bios Scientific Publishers
Limited, Oxford.
Davies, B.H. (1976) Carotenoids. Chemistry and
Biochemistry of Plant Pigments, 2nd edn, vol. 2 (ed. T.
W. Goodwin), pp. 38–165. Academic Press, London.
Demmig-Adams, B. & Adams III, W.W. (1992)
Photoprotection and other responses of plants to high
light stress. Annual Review of Plant Physiological and
Plant Molecular Biology 43, 599–626.
Ehleringer, J.R. & Cooper, T.A. (1992) On the role of orientation in reducing photoinhibitory damage in photosynthetic-twig
desert
shrubs.
Plant,
Cell
and
Environment 15, 301–306.
Feierabend, J. & Engel, S. (1986) Photoinactivation of catalase in vitro and in leaves. Archives of Biochemistry and
Biophysics 251, 567–576.
Feierabend, J., Schaan, C. & Hertwig, B. (1992) Photoinactivation of catalase occurs under both high- and lowtemperature stress conditions and accompanies photoinhibition of PSII. Plant Physiology 100, 1554–1561.
Foyer, C.H., Lelandais, M. & Kunert, K.J. (1994)
Photooxidative
stress
in
plants.
Physiologia
Plantarum 92, 696–717.
Freitas, H. & Breckle, S.W. (1992) Importance of bladder
hairs for salt tolerance of field-grown Atriplex species
from a Portuguese salt marsh. Flora 187, 283–297.
Hager, A. & Meyer-Bertenrath, T. (1966) Die Isolierung und
quantitative Bestimmung der Carotinoide und
Chlorophylle von Blättern, Algen und isolierten
Chloroplasten mit Hilfe dünnschichtchromatographischer
Methoden. Planta 69, 198–217.
Heber, U., Bligny, R., Streb, P. & Douce, R. (1996)
Photorespiration is essential for the protection of the photosynthetic apparatus of C3 plants against photoinactivation under sunlight. Botanica Acta 109, 307–315.
Hertwig, B., Streb, P. & Feierabend, J. (1992) Light dependence of catalase synthesis and degradation in leaves and
the influence of interfering stress conditions. Plant
Physiology 100, 1547–1553.
Krause, G.H. & Weis, E. (1991) Chlorophyll fluorescence
and photosynthesis: the basics. Annual Review of Plant
Physiology and Molecular Biology 42, 313–349.
Lang, M. & Schindler, C. (1994) The effect of leaf-hairs on
blue and red fluorescence emission and on zeaxanthin
cycle performance of Senecio medley L. Journal of Plant
Physiology 144, 680–685.
Ludlow, M.M. (1987) Light stress at high temperature.
Photoinhibition (eds D. J. Kyle, C. B. Osmond & C. J.
Arntzen), pp. 89–109. Elsevier Science Publishers,
Amsterdam.
Mishra, R.K. & Singhal, G.S. (1992) Function of photosynthetic apparatus of intact wheat leaves under high light
and heat stress and its relationship with peroxidation of
thylakoid lipids. Plant Physiology 98, 1–6.
Mittler, R., Nir, M. & Tel-Or, E. (1991) Antiperoxidative
enzymes in Retama and their seasonal variation. Free
Radical Research Communications 14, 17–24.
Mooney, H.A., Ehleringer, J. & Björkman, O. (1977) The
energy balance of leaves of the evergreen desert shrub
Atriplex hymenelytra. Oecologia 29, 301–310.
Moran, J.F., Becana, M., Iturbe-Ormaetxe, I., Frechilla, S.,
Klucas, R.V. & Aparicio-Tejo, P. (1994) Drought induces
oxidative stress in pea plants. Planta 194, 346–352.
Osmond, C.B., Björkman, O. & Anderson, D.J. (1980)
Physiological Processes in Plant Ecology. Toward a
Synthesis with Atriplex. Springer Verlag, Berlin.
Quartacci, M.F. & Navari-Izzo, F. (1992) Water stress and
free radical mediated changes in sunflower seedlings.
Journal of Plant Physiology 139, 621–625.
Robinson, S.A., Lovelock, C.E. & Osmond, C.B. (1993)
Wax as a mechanism for protection against photoinhibition—a study of Cotyledon orbiculata. Botanica
Acta 106, 307–312.
Sgherri, C.L.M. & Navari-Izzo, F. (1995) Sunflower
seedlings subjected to increasing water deficit stress:
oxidative stress and defence mechanisms. Physiologia
Plantarum 93, 25–30.
Sinclair, R. & Thomas, D.A. (1970) Optical properties of
leaves of some species in arid south Australia. Australian
Journal of Botany 18, 261–273.
Stocker, O. (1974a) Der Wasser- und Photosynthesehaushalt
von Wüstenpflanzen der südalgerischen Sahara. I.
Standorte und Versuchspflanzen. Flora 163, 46–88.
424
P. Streb et al.
© 1997 British
Ecological Society,
Functional Ecology,
11, 416–424
Stocker, O. (1974b) Der Wasser- und Photosynthesehaushalt
von Wüstenpflanzen der südalgerischen Sahara. III.
Jahresgang und Konstitutionstypen. Flora 163, 480–529.
Streb, P. & Feierabend, J. (1996) Oxidative stress responses
accompanying photoinactivation of catalase in NaCltreated rye leaves. Botanica Acta 109, 125–132.
Streb, P., Michael-Knauf, A. & Feierabend, J. (1993)
Preferential photoinactivation of catalase and photoinhibition of photosystem II are common early symptoms
under various osmotic and chemical stress conditions.
Physiologia Plantarum 88, 590–598.
Thayer, S.S. & Björkman, O. (1990) Leaf xanthophyll con-
tent and composition in sun and shade determined by
HPLC. Photosynthesis Research 23, 331–343.
Vierling, E. (1991) The roles of heat shock proteins in
plants. Annual Review of Plant Physiology and Molecular
Biology 42, 579–620.
Volk, S. & Feierabend, J. (1989) Photoinactivation of catalase at low temperature and its relevance to photosynthetic and peroxide metabolism in leaves. Plant, Cell and
Environment 12, 701–712.
Received 9 April 1996; revised 10 September 1996;
accepted 18 September 1996