Plant Science 152 (2000) 153 – 163
www.elsevier.com/locate/plantsci
Effect of acclimation to suboptimal temperature on
chilling-induced photodamage: comparison between a domestic
and a high-altitude wild Lycopersicon species
Jan Henk Venema a,*, Leen Villerius b, Philip R. van Hasselt a
a
b
Department of Plant Biology, Uni6ersity of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands
Department of Marine Biology, Uni6ersity of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands
Received 6 August 1999; received in revised form 11 October 1999; accepted 2 November 1999
Abstract
Plants of a domestic (Lycopersicon esculentum [L.] Mill. cv. Abunda) and a high-altitude wild Lycopersicon species (L.
peru6ianum Mill. LA 385) were grown at near-optimal (25/20°C) or suboptimal (16/14°C) temperature. Leaf discs from just fully
expanded leaves were exposed to an irradiance of 1000 mmol m − 2 s − 1 at 5°C for 48 h. The effect of growth temperature on the
susceptibility to photoinhibition of photosystem II (PSII) and its recovery, degradation of leaf pigments, chlorophyll (Chl)
fluorescence quenching and xanthophyll cycle activity were examined. Leaves of L. esculentum and L. peru6ianum plants grown
at optimal temperature, were similarly susceptible to photodamage. Suboptimal-grown leaves of both species showed a higher
tolerance to photoinhibition than optimal-grown leaves. In both species, recovery of photoinhibited PSII was more complete in
leaves grown at suboptimal than at optimal temperature. In contrast to L. esculentum, suboptimal-grown leaves of L. peru6ianum
exhibited faster kinetics of recovery from photoinhibition than optimal-grown leaves. Light-induced degradation of leaf pigments
in leaves grown at 16/14°C was 2.3- and 2.7-times slower in L. esculentum and L. peru6ianum, respectively, when compared with
leaves grown at 25/20°C. Non-photochemical quenching (NPQ) of Chl fluorescence developed faster in leaves of suboptimalgrown plants, and steady-state levels were 20% higher than in leaves of optimal-grown plants of both species. An increased pool
size of xanthophyll cycle pigments together with a slightly higher conversion state, resulted in a 1.5- (L. esculentum) or 3-fold (L.
peru6ianum) higher maximal zeaxanthin content in suboptimal-, as compared with optimal-grown leaves. These results demonstrate that acclimation to suboptimal temperature increased the capacity to resist chilling-induced photodamage in both the
domestic and the high-altitude wild Lycopersicon species. However, the acclimatory response was more pronounced in L.
peru6ianum than in L. esculentum, indicating a greater ability of the high-altitude wild species to acclimate its photosynthetic
apparatus to suboptimal temperatures. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Chlorophyll a fluorescence; Lycopersicon esculentum; Lycopersicon peru6ianum; Photodamage; Suboptimal temperature acclimation;
Xanthophyll cycle
1. Introduction
Abbre6iations: Fo, Fm and Fv, minimum, maximum and variable
Chl fluorescence where Fv = Fm –Fo; Fv/Fm, maximum quantum efficiency of PSII; F %v/F %m, quantum efficiency of open PSII reaction
centres; NPQ, non-photochemical quenching; PAR, photosynthetically active radiation; QA, primary stable electron acceptor of PSII
reaction centre; qP, photochemical quenching; FPSII, quantum yield of
PSII electron transport; SVAZ, total xanthophyll cycle pool.
* Corresponding author. Tel.: + 31-50-3632289; fax: + 31-503632273.
E-mail address: [email protected] (J.H. Venema)
The combination of low temperature and high
light enhances the induction of photodamage in
chilling-sensitive plants [1]. This is caused by the
low-temperature-induced inhibition of (i) photosynthetic capacity, (ii) repair mechanisms, (iii)
xanthophyll cycle-related energy dissipation, and
(iv) antioxidant enzyme activity [2]. The term
photodamage includes photoinhibition, defined as
the reversible decrease of photosynthetic efficiency
0168-9452/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 ( 9 9 ) 0 0 2 2 8 - 9
154
J.H. Venema et al. / Plant Science 152 (2000) 153–163
and capacity, as well as photooxidation, known as
the irreversible oxygen-dependent degradation of
components of the chloroplast including photosynthetic pigments [3]. The site of photoinhibition
is generally considered to be located in photosystem (PS) II, however, in chilling-sensitive cucumber (Cucumis sati6us), it was shown that PSI is the
main site of damage during chilling under weak
illumination (for a review, see [4]).
Investigations with chilling-resistant species like
spinach (Spinacia oleracea) [5,6], potato (Solanum
tuberosum and S. commersonii ) [7] and winter
cereals [8] showed that low-temperature acclimation increased the resistance to chill-induced photoinhibition. Up to now, it was considered that
growth at low temperature was an absolute requirement for the acquisition of this tolerance to
high light [9], however recently, it was observed
that increased photoprotection capacity can be
obtained by a long-term temperature shift [10].
Several mechanisms as (i) increased photosynthetic
capacity [11], (ii) increased amounts and activities
of active oxygen scavengers [12], (iii) increased
capacity for violaxanthin de-epoxidation and related dissipation of excess energy [10,12–15], or a
combination of these mechanisms [16], are considered to be involved in the reduced susceptibility to
photoinhibition of PSII at chilling temperatures
after cold acclimation. In contrast to chilling-tolerant species, experiments with maize (Zea mays)
suggested that chilling-sensitive plants have a low
ability to increase tolerance to chill-induced photoinhibition after acclimation to low temperature
[17–20].
Comparison of the susceptibility of the domestic
tomato (Lycopersicon esculentum) and high-altitude ecotypes of the wild relative L. hirsutum to
long-term chilling under photoinhibitory light conditions, demonstrated a comparable decline, but
faster recovery of light-saturated photosynthetic
CO2-uptake in leaves of L. hirsutum [21]. In the
high-altitude species L. peru6ianum LA 385,
growth and photosynthesis were less affected by
long-term chilling in comparison to L. esculentum
cv. Abunda [22,23]. Evidence was presented that
both L. hirsutum [24] and L. peru6ianum [25] have
neither inherently higher antioxidant levels nor
distinct activities of enzymes in the ascorbate/glutathione cycle than L. esculentum leaves, with the
exception of a higher glutathione reductase activity in the wild species. So far, all mentioned stud-
ies with L. peru6ianum were performed under low
to moderate irradiance levels which were non-photoinhibitory. In the present study, we analyzed for
the first time the sensitivity of L. peru6ianum and
L. esculentum to high light during chilling in order
to compare the ability of both species to tolerate
light-induced stress. In addition, we studied the
impact of acclimation to suboptimal temperature
on chill-induced photodamage since it was shown
that both species showed differential photosynthetic acclimation capacities to suboptimal temperature [26].
The objectives of the present study were (1) to
compare the susceptibility of the chilling-sensitive
domestic tomato (L. esculentum) and the more
chilling-tolerant high-altitude wild relative L. peru6ianum to chilling under high light and (2), to
investigate the impact of suboptimal temperature
acclimation on the susceptibility to photodamage
in both species. Photodamage was generated by
exposing leaf discs from just fully expanded leaves
of plants grown at either optimal or suboptimal
temperature, to severe chilling (5°C) in the presence of a high PAR (1000 mmol m − 2 s − 1) as
applied earlier in investigations with cucumber (C.
sati6us) [27], vine (Vitis 6inifera) [28] and maize
[20]. The time courses of photoinhibition, recovery
and pigment degradation were analyzed in relation
with the operation of the xanthophyll cycle and
development of non-photochemical quenching
(NPQ) of chorophyll (Chl) a fluorescence.
2. Materials and methods
2.1. Plant material and growth conditions
The Lycopersicon species investigated in this
study were the domestic tomato L. esculentum (L.)
Mill. cv. Abunda and the more cold-tolerant,
high-altitude wild species L. peru6ianum Mill. LA
385 (originating from San Juan, Prov. Cajamarca,
Peru, 2400 m at sea level). Seeds of L. esculentum
cv. Abunda were obtained from Rijk Zwaan, De
Lier, The Netherlands, whereas seeds of L. peru6ianum were provided by the DLO-Centre for Plant
Breeding and Reproduction research (CPRODLO), Wageningen, The Netherlands. Plants were
grown from seed in 1-l potting soil (Efbe, F. Bos
B.V., Heiligerlee, The Netherlands) in a greenhouse at 25/20°C, 14/10 h day/night regime with
J.H. Venema et al. / Plant Science 152 (2000) 153–163
supplementary light (Philips HPI-T 400 W) at a
minimum PAR of 225 mmol m − 2 s − 1 (measured
with a quantum sensor, SKP 215, Skye, Llandrindod Wells, UK). At a plastochron index of
5, plants were randomly distributed between
two controlled-environment cabinets (Conviron
EF7H, Winnipeg, Canada) at 25/20°C (‘optimal’),
or at 16/14°C (‘suboptimal’) day/night temperature. In both cabinets, a PAR of 225 9 25 mmol
m − 2 s − 1 during a 12 h photoperiod was provided
for 95% by fluorescent lamps (Sylvania F72T12/
CW/VHO) and for 5% by incandescent lamps
(Sylvania 60 W). The relative humidity in both
cabinets was 559 10%.
2.2. High-light treatment at chilling temperature
Leaf discs (11 mm diameter) were punched from
the youngest mature leaves of plants which were
grown at either the optimal or suboptimal temperature, and irradiated with white light of 1000 mmol
m − 2 s − 1, provided by two 400 W lamps (Philips
HPI PLUS) in a cold room at 5°C. During illumination, leaf discs floated on tap water in a home
build leaf cuvette with a Plexiglas lid in which the
temperature was kept at 5°C by circulating water
from a cooling bath. To prevent heating of the
samples, light was filtered through a water layer of
10 cm.
2.3. Chl a fluorescence measurements
Susceptibility to photoinhibition was analyzed
as the time-dependent loss of photosynthetic efficiency judged from changes in the ratio of variable
to maximal Chl fluorescence (Fv/Fm) [29]. After
illumination at 5°C, leaf discs were dark adapted
for 15 min at room temperature after which the
Fv/Fm ratio was measured with a pulse amplitude
modulation fluorometer (PAM-2000, Heinz Walz
GmbH, Effeltrich, Germany). For subsequent recovery, leaf discs were placed on a water layer at
room temperature and illuminated with low white
light of 25 mmol m − 2 s − 1.
The development of thermal energy dissipation
in PSII (NPQ) and steady-state Chl a fluorescence
characteristics, were measured using the PAM101/-103 (Heinz Walz GmbH, Effeltrich, Germany). Two Schott lamps (KL 1500, Mainz,
Germany) provided saturating light flashes and
actinic illumination. The measurements were per-
155
formed with leaf discs (11 mm diameter) in a
temperature-controlled leaf cuvette (LD2, Hansatech, UK) at 25°C (optimal-grown leaves) or at
16°C (suboptimal-grown leaves), and at 5°C. During the entire measurement, a slow stream of
humidified air was passed over the leaf samples.
The maximum fluorescence levels Fm (darkadapted leaves) and F %m (light-adapted leaves) were
determined by application of saturating flashes (1
s) of 3500 mmol m − 2 s − 1. Steady-state fluorescence (F) was measured at actinic irradiances of
225 and 1000 mmol m − 2 s − 1 that were similar to
the PAR during growth and the high-light treatment, respectively. The minimum fluorescence
level (Fo) was sensitized by an irradiance of 0.1
mmol m − 2 s − 1 at a modulation of 1.6 kHz in a
far-red background light. F %o was measured directly after the actinic light phase by illumination
with far-red light (50 mmol m − 2 s − 1) in order to
account for Fo quenching. Chl a fluorescence
parameters were calculated as described in Venema et al. [26].
2.4. Pigment determination
Within 3 s after illumination, leaf discs were
immersed in liquid N2 and stored at −80°C until
use. The frozen leaf discs were ground to powder
in liquid N2 with 2 mg of solid Na2CO3. Pigments were extracted with 1 ml ice-cold acetone.
After centrifugation for 15 s in a microfuge, pigments were separated by HPLC. The HPLC system was a Pharmacia-LKB liquid chromatograph
(Uppsala, Sweden) consisting of two high-pressure
pumps (model 2150), a LC-controller (model
2252), a cooled (2–3°C) autosampler (model 2157)
and a two-channel variable wavelength detector
(model 2141). The method used was a modification of that, described by Gilmore and Yamamoto
[30]. A Waters Delta-Pak (Milford, MA) reversedphase column (5 mm, C18, 100 A, , 3.9 × 150 mm2,
17% carbon load, fully end capped) and a Waters
Nova-Pak guard column (5 mm, C18) were used.
Column temperature was maintained at 15°C by a
column thermostat (model BFO-0415, W.O. Electronika, Langenzersdorf, Germany). The binary
solvent system consisted of solvent A, acetonitrile:methanol:Tris –HCl (0.2 M, pH 8.0)
(60:20:20, v/v/v) and solvent B, acetonitrile:ethanol:ethylacetate (50:30:20, v/v/v). Prior to
injection, the column was equilibrated for 5 min
J.H. Venema et al. / Plant Science 152 (2000) 153–163
156
by flushing a mixture of 90% mobile phase A and
10% of mobile phase B. Finally, the column was
re-equilibrated for 2 min with a mixture of 90%
Table 1
Maximum PSII quantum efficiency (Fv/Fm) of dark-adapted
leaves and the relative reduction state of PSII (1−qP) and
non-photochemical quenching (NPQ)a in the youngest mature
leaves of L. esculentum and L. peru6ianum grown at optimal
(25/20°C) or suboptimal (16/14°C) temperatureb
Parameter L. esculentum
Fv/Fm
1−qP
NPQ
L. peru6ianum
25/20°C
16/14°C
25/20°C
16/14°C
0.82
90.01
0.14
90.01
0.65
9 0.21
0.81
90.01
0.31
90.05**
1.23
90.25*
0.82
90.01
0.09
90.01
0.23
90.05
0.83
90.01
0.12
90.02
0.54
90.11*
a
qP and NPQ were measured at a temperature (25 or 16°C)
and actinic irradiance level (225 mmol photons m−2 s−1)
which were equal to the growth conditions.
b
Data represent means of three individual plants 9 S.D.
* Significantly different pairwise comparisons between
treatments (Student t-test) are indicated. P50.05.
** Significantly different pairwise comparisons between
treatments (Student t-test) are indicated. P50.01.
Table 2
Steady-state Chl a fluorescence characteristics under 1000
mmol m−2 s−1 at 5°C in the youngest mature leaves of L.
esculentum cv. Abunda and L. peru6ianum LA 385 grown at
optimal (25/20°C) or suboptimal (16/14°C) temperaturea
Parameter L. esculentum
qP
F %v/F %m
FPSII
NPQ
L. peru6ianum
25/20°C
16/14°C
25/20°C
16/14°C
0.04
90.01
0.37
90.03
0.01
90.01
2.07
90.09
0.07
90.02
0.41
90.03
0.03
9 0.01
2.43
9 0.11*
0.06
90.02
0.46
9 0.04
0.02
9 0.01
1.93
90.07
0.10
90.03
0.44
90.03
0.04
90.01
2.52
90.09***
Data represent means of three individual plants 9S.D.
* Significantly different pairwise comparisons between
treatments (Student t-test). P50.05.
*** Significantly different pairwise comparisons between
treatments (Student t-test). P50.001.
a
solvent A and 10% solvent B, prior the next
injection. The flow rate was 1.0 ml min − 1, the
maximum back-pressure 74 bar. The pigments
were detected at 436 nm. Data were recorded
automatically using Nelson 2600 chromatografic
software (Perkin–Elmer Ltd., Beaconsfield, UK).
Pigments were identified as based upon their retention times relative to known standards and by
their absorption spectra, measured with a Cary
3E spectrophotometer. Concentrations were calculated as based on calibration-curves created
with the standards.
3. Results
3.1. Chl fluorescence characteristics
Leaves of both Lycopersicon species showed no
photoinhibition during growth at the suboptimal
temperature regime as indicated by the unaffected
Fv/Fm values (Table 1). The excitation pressure
experienced by the leaves, measured as the relative reduction state of PSII (1 −qP), only increased in L. esculentum in response to
suboptimal temperature. NPQ was about 2-fold
higher at suboptimal temperature in both species.
Remarkably, L. esculentum showed significantly
higher NPQ levels than L. peru6ianum at both
temperature regimes.
Steady-state Chl fluorescence characteristics, qP,
F %v/F %m and FPSII, did not differ significantly between optimal- and suboptimal-grown leaves in
both species (Table 2) subjected to high-light
chilling conditions. In contrast, steady-state NPQ
levels were 20% higher in leaves of both Lycopersicon species grown at suboptimal temperature. In Fig. 1, the increase in NPQ at the start
of the high-light chilling treatment is shown for
both leaf types. The development of NPQ was
faster in suboptimal- than in optimal-grown
leaves of both species. Significantly higher NPQ
levels were already apparent after 3 min in both
L. esculentum, viz 1.4-fold (PB0.01), and L. peru6ianum, viz 1.3-fold (PB0.05). Irrespective of
the growth temperature, NPQ development was
slightly faster in L. esculentum than in L. peru6ianum leaves, however, steady-state NPQ levels
were similar in both species.
J.H. Venema et al. / Plant Science 152 (2000) 153–163
Fig. 1. Development of thermal energy dissipation of PSII
(NPQ) during illumination with 1000 mmol m − 2 s − 1 at 5°C
in the youngest mature leaves of L. esculentum cv. Abunda
(A) and L. peru6ianum LA 385 (B) grown at optimal () or
suboptimal () temperature. Data represent means 9S.D. of
three individual plants.
157
values in the domestic and wild species grown at
optimal temperature, viz from 0.82090.010 to
0.11890.026 in L. esculentum and to 0.129 9
0.045 in L. peru6ianum. The decline in Fv/Fm was
slower in leaves of plants grown at suboptimal
than at optimal temperature (Fig. 2A, C). This
acclimative response was more pronounced for L.
peru6ianum than for L. esculentum (Table 3). Also,
in both species, recovery of Fv/Fm was more complete in suboptimal- than in optimal-leaves (Fig.
2B, D). First-order rate constants of recovery,
analyzed during the first 2 h under low light at
20°C, showed that in L. peru6ianum Fv/Fm recovered significantly (PB0.05) faster in suboptimalgrown leaves (0.21590.025 h − 1) than in
optimal-grown leaves (0.140 90.019 h − 1), whereas
in L. esculentum, recovery rates of Fv/Fm were
comparable in leaves of optimal- (0.07890.019
h − 1) and suboptimal-grown plants (0.10490.013
h − 1).
3.3. Pigment composition
Significantly (PB0.05) higher contents of Chl a,
Chl b and all carotenoids were measured on a leaf
area basis in suboptimal-leaves of L. peru6ianum
whereas in L. esculentum only the size of the
xanthophyll cycle pool (SVAZ) was increased at
16/14°C (see 0 h points in Figs. 3 and 4). The
increase in SVAZ in response to suboptimalgrowth temperature was considerably larger in L.
Fig. 2. Time courses of photoinhibition and recovery in the
youngest mature leaves of L. esculentum cv. Abunda (A, C)
and L. peru6ianum LA 385 (B, D) as analyzed by changes in
the maximum quantum efficiency of PSII photochemistry
(Fv/Fm). The photoinhibition treatments were performed for 5
h (optimal-grown leaves, ) or 7 h (suboptimal-grown leaves,
) at 1000 mmol m − 2 s − 1 and 5°C. Subsequent recovery was
followed for 17 h (optimal-grown leaves, ) or 15 h (suboptimal-grown leaves, ) at 20°C under 25 mmol m − 2 s − 1. Data
represent means 9S.D. of six leaf discs, taken from three
individual plants. If not shown, S.D. is smaller than symbol.
3.2. Photoinhibition of PSII and subsequent
reco6ery
Exposure of leaf discs to high light at 5°C
during 5 h, resulted in a similar drop in Fv/Fm
Table 3
Impact of growth temperature on the time (h) required to
decrease 50% of the initial Fv/Fm value and pigment content
(t1/2) during illumination with 1000 mmol m−2 s−1 at 5°C in
the youngest mature leaves of L. esculentum cv. Abunda and
L. peru6ianum LA 385 grown at optimal (25/20°C) or suboptimal (16/14°C) temperaturea
Parameter
Fv/Fm
Chl a
Chl b
Neoxanthin
Lutein
b-carotene
SVAZ
a
t1/2 (h)
L. esculentum
L. peru6ianum
25/20°C
16/14°C
25/20°C
16/14°C
2.2
14
14
10
12
9
8
3.5
35
35
23
21
21
19
2.3
17
16
12
15
10
10
4.7
\48
43
34
36
26
31
Data represent means of three individual plants.
158
J.H. Venema et al. / Plant Science 152 (2000) 153–163
Fig. 3. Degradation of Chl a (
, ) and Chl b (, ) (A, E), neoxanthin (B, F), lutein (C, G) and b-carotene (D, H) during
illumination with 1000 mmol m − 2 s − 1 at 5°C in the youngest mature leaves of L. esculentum cv. Abunda and L. peru6ianum LA
385 grown at optimal (closed symbols) or suboptimal (open symbols) temperature. Data represent means 9S.D. of three
individual plants. If not shown, S.D. is smaller than symbol.
peru6ianum (95%) than in L. esculentum (50%).
Calculated on a Chl basis, the increase in SVAZ
was 103 and 42% in L. peru6ianum and L. esculentum, respectively, whereas the neoxanthin content
was higher only in L. peru6ianum (data not
shown). b-carotene and lutein contents were unaffected by the growth temperature in both species
when related to the Chl content. In both species,
the conversion state of the xanthophyll cycle pool
(A+ Z)/(V +A +Z) was slightly higher in suboptimal-leaves at the start of the high-light treatment
(Fig. 5), due to a higher content of antheraxanthin
(Fig. 4B, F).
3.4. Xanthophyll cycle acti6ity and pigment
degradation
In spite of the low temperature, violaxanthin
was rapidly de-epoxidized to antheraxanthin and
zeaxanthin (Fig. 4). The formation of zeaxanthin
was slowest in leaves of optimal-grown L. peru6ianum (Fig. 4G). After a lag phase of 6 h, during
which the Fv/Fm ratio decreased to very low values
(Fig. 3A, C), the degradation of leaf pigments
started. In both Lycopersicon species, the rate of
pigment degradation was clearly affected by the
growth temperature: leaves of suboptimal-grown
J.H. Venema et al. / Plant Science 152 (2000) 153–163
159
Fig. 4. Changes in the content of violaxanthin (A, E), antheraxanthin (B, F), zeaxanthin (C, G) and the total xanthophyll cycle
pool size (D, H) during illumination with 1000 mmol m − 2 s − 1 at 5°C in the youngest mature leaves of L. esculentum cv. Abunda
and L. peru6ianum LA 385 grown at optimal (
) or suboptimal () temperature. Data represent means 9 S.D. of three
individual plants. If not shown, S.D. is smaller than symbol.
Fig. 5. Changes in the xanthophyll cycle conversion state (A + Z)/(V+ A+ Z) during illumination with 1000 mmol m − 2 s − 1 at
5°C in the youngest mature leaves of L. esculentum cv. Abunda (A) and L. peru6ianum LA 385 (B) grown at optimal (
) or
suboptimal () temperature. Data represent means 9S.D. of three individual plants. If not shown, S.D. is smaller than symbol.
160
J.H. Venema et al. / Plant Science 152 (2000) 153–163
plants showed about a 2.3- (L. esculentum) or
2.7-times (L. peru6ianum) slower degradation of all
pigments compared with leaves of optimal-grown
plants (Table 3). The time required for degradation
of 50% of the different leaf pigments (t1/2) strongly
varied. The data clearly show that carotenoids are
more susceptible for photodamage than the chlorophylls. Within the group of carotenoids, pigments
of the xanthophyll cycle and b-carotene are generally faster degradated than neoxanthin and lutein.
In both species, (A+ Z)/(V + A+ Z) rapidly increased during the 1st hour in high light at 5°C
(Fig. 5). (A+Z)/(V +A+ Z) reached slightly
higher values in leaves of suboptimal- (0.70–0.74)
than of optimal-grown plants (0.57–0.65). Due to
a pronounced slower degradation of xanthophyll
cycle pigments in suboptimal than in optimal
leaves, (A+ Z)/(V +A + Z) kept a stable high level
for more than 36 h in suboptimal leaves, whereas
in optimal leaves, (A + Z)/(V + A+ Z) decreased
rapidly after reaching its maximum value.
4. Discussion
4.1. Inter-specific comparison of PSII
photoinhibition susceptibility
Although L. esculentum has been reported to be
more sensitive to long-term chilling under low light
than L. peru6ianum [22,23], the present study
showed that optimal-grown leaves of both species
were similarly susceptible to photoinhibition of
PSII under high light at 5°C (Fig. 2A, C). This
result is in accordance with the observation that
the decrease of light-saturated photosynthetic CO2
uptake in leaves of a high-altitude ecotype of the
wild species L. hirsutum was comparable with the
decrease in L. esculentum leaves during a 7-day
chilling treatment under 400 mmol m − 2 s − 1 at
10°C [21]. Comparisons between altitudinal ecotypes of L. hirsutum also revealed similar susceptibility to photoinhibition [31], whereas in another
study [32], a high-altitude ecotype showed only
slightly higher resistance to chill-induced photoinhibition than an ecotype originating from low
altitude. Taken together, these observations
strongly suggest that the altitudinal distribution of
Lycopersicon species is not determined by differences in susceptibility to chill-induced photoinhibition of PSII. Tolerance of cereals to short-term
photoinhibition (hours) was reflected by the capac-
ity to keep the primary electron carrier of PSII, QA,
in the oxidized state [9]. The relative oxidation
state of QA, as estimated by qP (Table 2), did not
differ between leaves of optimal-grown L. esculentum and L. peru6ianum plants under the present
high-light chilling conditions, confirming their similar susceptibility to PSII photoinhibition. It
should be stressed that PSII might not be the sole
site of photoinhibition in tomato leaves at low
temperature. Under either weak illumination (0–
220 mmol m − 2 s − 1) in chilling-sensitive plants [4]
or at high irradiance (900 and 3700 mmol m − 2 s − 1)
in chilling-resistant potato leaves [33], PSI was
shown to be the primary target for photoinhibition. For chilling-sensitive plants, however, weak
illumination is essential for selective photoinhibition of PSI whereas stronger illumination, as applied in this study, induces photoinhibition of PSII
[4,34]. In agreement, the decrease in photosynthetic
oxygen evolution measured at low irradiance during chilling of L. esculentum leaves at high light,
was related to photoinhibition of PSII as judged
from Fv/Fm measurements [25]. In addition, it has
been shown that short-term chilling of L. esculentum at high light also inhibits the reductive activation of regulatory enzymes of the Calvin cycle
[35,36].
4.2. Xanthophyll cycle-related energy dissipation
It is assumed that the xanthophyll cycle-related
quenching of absorbed excitation energy in the
pigment bed of PSII plays an important role in
photoprotection of the photosynthetic apparatus
(for reviews cf. [37,38]. However, the rate of deepoxidation of violaxanthin to zeaxanthin via antheraxanthin, and related development of NPQ, is
strongly inhibited at low temperatures [39–41].
Nevertheless, in both examined Lycopersicon species 90% of the maximum convertible violaxanthin was de-epoxidized after 1 h of illumination at
5°C (Fig. 4), whereas steady-state levels of NPQ
were reached in 30 min (Fig. 1). Comparison
between chilling-tolerant and chilling-sensitive species demonstrated that the conversion rate of xanthophyll cycle pigments in the chilling-tolerant
species was less affected by low temperature
[14,40]. Our data demonstrate that in more closely
related optimal-grown Lycopersicon species, with
similar susceptibility to chill-induced photoinhibition, the xanthophyll cycle activity and NPQ capacity were comparable at 5°C (Figs. 1 and 4).
J.H. Venema et al. / Plant Science 152 (2000) 153–163
Comparison of altitudinal ecotypes of L. hirsutum
showed that a high-altitude ecotype, with a slightly
higher photoinhibition tolerance, accumulated also
slightly more zeaxanthin than a low-altitude ecotype during short-term chilling under high light
[32]. In contrast, no significant differences appeared in the operation of the xanthophyll cycle at
chilling temperatures between maize genotypes differing in chilling tolerance of the photosynthetic
apparatus [42]. These and our data suggest that the
xanthophyll cycle activity at low temperatures is
similar within or between closely related species
and therefore probably not a major factor determining possible differences in the capacity of the
photosynthetic apparatus to cope with high-light
stress at low temperature.
4.3. Acclimatory effects on photodamage
susceptibility, reco6ery and xanthophyll cycle
In accordance with recent results [26], the higher
PSII excitation pressure (1− qP) in L. esculentum
than in L. peru6ianum leaves measured under suboptimal temperature conditions (Table 1), demonstrated a lower capacity of the domesticated
tomato in comparison with its wild relative L.
peru6ianum, to acclimate chloroplast functioning to
suboptimal temperature. Nevertheless, suboptimalgrown leaves of L. esculentum showed a considerably higher tolerance towards PSII photoinhibition
and photooxidation than optimal-grown leaves
(Table 3). The increased tolerance to PSII photoinhibition induced by growth at suboptimal temperature was more pronounced in both Lycopersicon
species in comparison with maize [17–20], demonstrating inter-specific variation of chilling-sensitive
species in their capacity for acclimation to suboptimal temperature. In both Lycopersicon species,
suboptimal-grown leaves showed no significantly
higher qP values under the high-light chilling conditions than optimal-grown leaves, implying that a
difference in the efficiency to convert light energy
to photochemistry was not the reason for the
observed higher tolerance of suboptimal-grown
leaves to photoinhibition of PSII. As demonstrated
for winter rye (Secale cereale) [43], it should be
mentioned that the susceptibility of PSI to photoinhibition might be decreased after growth at
lower temperatures as well.
In several studies, a strong correlation was
found between NPQ and the zeaxanthin content
[39–41]. Indeed, the faster development and the
161
higher steady-state level of NPQ observed in suboptimal leaves of both species was related to faster
de-epoxidation kinetics and to the formation of
higher zeaxanthin and antheraxanthin levels. The
formation of higher amounts of these de-epoxidized xanthophylls was associated with an increased xanthophyll cycle pool size (Fig. 4D, H),
and, to a small extent, to a higher conversion state
of the xanthophyll cycle (Fig. 5) in suboptimalgrown leaves. Similar effects of low-temperature
acclimation on the operation of the xanthophyll
cycle were monitored in other species [10,14–
17,22,40] and can therefore be regarded as a general acclimatory response of plants to facilitate a
higher level of photoprotection [38].
In contrast to L. esculentum, L. peru6ianum
exhibited a higher capacity for fast recovery of
Fv/Fm in suboptimal- than in optimal-grown leaves
(Fig. 2B, D). It was found that the fast recovery
phase of PSII photoinhibition was kinetically
closely related with the epoxidation of zeaxanthin
[15,44]. In these reports, it was speculated that
zeaxanthin could be involved in the formation of a
photoinhibitory quenching state, characterized by
energy-dissipating PSII with undamaged D1
protein. The higher zeaxanthin contents in suboptimal- than in optimal-grown leaves of both species
(Fig. 4C, G), may therefore indicate the presence
of more energy-dissipating PSII. Since zeaxanthin
contents in suboptimal-grown leaves of L. esculentum and L. peru6ianum were comparable after 7 h
of illumination at 5°C, the faster recovery of Fv/Fm
in suboptimal-grown L. peru6ianum suggests a
faster epoxidation of zeaxanthin during the first
hours of recovery in this wild species than in
suboptimal-grown L. esculentum.
It seems unlikely that the higher resistance to
photodamage of Lycopersicon leaves acclimated to
suboptimal temperature can completely be ascribed to the relative small increase (20%) in NPQ
capacity. Taking into account the low specific
activity in maize of four of the five reactive oxygen
scavenging enzymes at 5°C [45], and an imperfect
acclimatory adjustment in the activity of these
enzymes in response to suboptimal growth temperature [46], it seems improbable that the enzymatic
scavenging system plays a significant role in increasing the photoprotective capacity of suboptimal-grown leaves at low temperatures. More
relevant for the superior tolerance of suboptimalgrown Lycopersicon leaves against photodamage
162
J.H. Venema et al. / Plant Science 152 (2000) 153–163
may be the accumulation of non-enzymatic antioxidants (ascorbate, glutathione and a-tocopherol),
as was found in suboptimal-grown maize [20] and
cold-acclimated spinach leaves [12].
Summarizing, optimal-grown leaves of L. esculentum and L. peru6ianum were similarly susceptible to photodamage, and showed comparable
xanthophyll cycle activity and NPQ capacity during high-light chilling. Acclimation to suboptimal
temperature increased the tolerance to chill-induced photodamage in both species but the increase was more pronounced in the wild L.
peru6ianum species. The higher resistance towards
photodamage of suboptimal-grown leaves was associated with a faster kinetics and higher level of
xanthophyll cycle-related dissipation of excess energy which was related to a faster formation of
higher amounts of de-epoxidized xanthophylls.
Acknowledgements
We thank Professor P.J.C. Kuiper for valuable
comments on the manuscript. This research was
financially supported by the Life Sciences Foundation (SLW), part of the Netherlands Organization
for Scientific Research (NWO).
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