Journal of Experimental Botany, Vol. 54, No. 383, pp. 851±860, February 2003
DOI: 10.1093/jxb/erg080
RESEARCH PAPER
Salinity treatment shows no effects on photosystem II
photochemistry, but increases the resistance of
photosystem II to heat stress in halophyte Suaeda salsa
Congming Lu1,2,5, Nianwei Qiu1,2, Baoshan Wang3 and Jianhua Zhang4,5
1
Photosynthesis Research Centre, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, P.R. China
Key Lab of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of
Sciences, Beijing 100093, P.R. China
2
3
4
Department of Biology, Shandong Normal University, Jinan 250014, P.R. China
Department of Biology, Hong Kong Baptist University, Hong Kong, P.R. China
Received 6 August 2002; Accepted 11 October 2002
Abstract
Photosynthetic gas exchange, modulated chlorophyll
¯uorescence, rapid ¯uorescence induction kinetics,
and the polyphasic ¯uorescence transients were
used to evaluate PSII photochemsitry in the halophyte Suaeda salsa exposed to a combination of
high salinity (100±400 mM NaCl) and heat stress
(35±47.5 °C, air temperature). CO2 assimilation rate
increased slightly with increasing salt concentration
up to 300 mM NaCl and showed no decrease even at
400 mM NaCl. Salinity treatment showed neither
effects on the maximal ef®ciency of PSII photochemistry (Fv/Fm), the rapid ¯uorescence induction
kinetics, and the polyphasic ¯uorescence transients
in dark-adapted leaves, nor effects on the ef®ciency
of excitation energy capture by open PSII reaction
centres (Fv¢/Fm¢) and the actual PSII effciency (FPSII),
photochemical quenching (qP), and non-photochemical quenching (qN) in light-adapted leaves. The
results indicate that high salinity had no effects on
PSII photochemistry either in a dark-adapted state or
in a light-adapted state. With increasing temperature,
CO2 assimilation rate decreased signi®cantly and no
net CO2 assimilation was observed at 47.5 °C.
Salinity treatment had no effect on the response of
CO2 assimilation to high temperature when temperature was below 40 °C. At 45 °C, CO2 assimilation rate
in control plants decreased to zero, but the saltadapted plants still maintained some CO2 assimilation capacity. On the other hand, the responses of
PSII photochemistry to heat stress was modi®ed by
salinity treatment. When temperature was above
35 °C, the declines in Fv/Fm, FPSII, Fv¢/Fm¢, and qP
were smaller in salt-adapted leaves compared to control leaves. This increased thermostability was independent of the degree of salinity, since no signi®cant
changes in the above-described ¯uorescence parameters were observed among the plants treated with
different concentrations of NaCl. During heat stress,
a very clear K step as a speci®c indicator of damage
to the O2-evolving complex in the polyphasic ¯uorescence transients appeared in control plants, but did
not get pronounced in salt-adapted plants. In addition, a greater increase in the ratio (Fi±Fo)/(Fp±Fo)
which is an expression of the proportion of the QBnon-reducing PSII centres was observed in control
plants rather than in salt-adapted plants. The results
suggest that the increased thermostability of PSII
seems to be associated with the increased resistance
of the O2-evolving complex and the reaction centres
of PSII to high temperature.
Key words: Chlorophyll ¯uorescence, gas exchange, heat
stress, photosystem II photochemistry, salinity treatment,
Suaeda salsa L.
5
To whom correspondence should be addressed. E-mail: [email protected]; [email protected]
Abbreviations: Fo, minimal ¯uorescence level in dark-adapted leaves; Fo¢, minimal ¯uorescence level in light-adapted leaves; Fm, maximal ¯uorescence level
in dark-adapted leaves; Fm¢, maximal ¯uorescence level in light-adapted leaves; Fv, maximum variable ¯uorescence level in dark-adapted leaves; Fv¢,
maximum variable ¯uorescence level in light-adapted leaves; Fs, steady-state ¯uorescence level; Fv/Fm, maximal ef®ciency of PSII photochemistry; FPSII,
actual PSII ef®ciency; Fv¢/Fm¢, ef®ciency of excitation energy capture by open PSII reaction centres; qP, photochemical quenching coef®cient; qN, nonphotochemical quenching coef®cient.
Journal of Experimental Botany, Vol. 54, No. 383, ã Society for Experimental Biology 2003; all rights reserved
852 Lu et al.
Introduction
The growth of plants is ultimately reduced by salinity
stress although plant species differ in their tolerance to
salinity (for a review, see Munns and Termaat, 1986). The
decline in growth observed in many plants subjected to
salinity stress is often associated with a decrease in their
photosynthetic capacity. Although much effort has been
invested into investigating the cause of decreased photosynthetic capacity, the underlying mechanisms are still
unclear. Since photosystem II (PSII) is believed to play a
key role in the response of photosynthesis to environmental perturbations (Baker, 1991), the effects of salinity stress
on PSII have been investigated extensively. However, the
collected data on the effects of salinity stress on PSII
photochemistry are con¯icting. Some studies have shown
that salt stress inhibits PSII activity (Bongi and Loreto,
1989; Mishra et al., 1991; Masojidek and Hall, 1992;
Belkhodja et al., 1994; Everard et al., 1994), whereas other
studies have indicated that salt stress has no effect on PSII
(Robinson et al., 1983; Brugnoli and BjoÈrkman, 1992;
Morales et al., 1992; AbadõÂa et al., 1999).
Under natural conditions, multiple environmental
stresses co-occur frequently. Thus, plants grown under
natural conditions are often subjected to the interaction of
multiple environmental stresses. It has been reported that
the responses of plants to several simultaneous stresses are
complex and usually not predictable by single-factor
analyses and that a combination of different environmental
stress factors can result in intensi®cation, overlapping or
antagonistic effects (Osmond et al., 1986). The effects of
interaction of salinity stress and high light on PSII have
been well investigated. Many studies have shown that
salinity stress predisposes PSII photoinhibitory damage
(Mishra et al., 1991; Masojidek and Hall, 1992). However,
much less attention has been paid to the responses of PSII
to a combination of salinity and heat stress. It is obviously
important to study the combination of these two stresses
from an ecophysiological viewpoint since about one-third
of the irrigated land worldwide has already been plagued
by increasing salinity (Epstein et al., 1980), particularly in
arid and semi-arid regions where salinity stress is often
combined with heat stress.
The Chenopodiaceae Suaeda salsa L., a C3 plant, is one
of the most important halophytes in China (Wang et al.,
2001) and has important economical values because its
seeds contain c. 40% oil, rich in unsaturated fatty acids,
which can be easily converted to chemical compounds for
industrial use (Zhao, 1998). S. salsa is native to saline soils
and adapted to the high salinity region in the north of China
where the temperature in the summer is as high as 45 °C.
Thus, S. salsa is exposed not only to salinity stress but also
to high temperature.
The objectives of this study were (1) to evaluate whether
salinity treatment affects PSII photochemistry, and (2) to
investigate how PSII responds to high temperature in saltadapted plants. To these ends, the changes in PSII
photochemistry and its thermostability in salt-adapted S.
salsa were examined by analysis of ¯uorescence quenching, rapid ¯uorescence induction kinetics (i.e. Kautsky
curve), and the polyphasic rise of ¯uorescence transients,
which have been shown to be useful, reliable, and noninvasive methods to assess PSII functions (Krause and
Weis, 1991; Schreiber et al., 1994; Govindjee, 1995;
Strasser et al., 1995; Strasser, 1997). The major results of
this study showed that salinity treatment had no effect on
PSII photochemistry, but increased thermostability of
PSII, which may be associated with increased thermostability of the whole PSII, such as the O2-evolving
complex and the reaction centre.
Materials and methods
Plant material and heat stress treatments
The seedlings of Suaeda salsa L. were grown in plastic pots (14 cm in
diameter and 13 cm in height) ®lled with sand and watered daily with
half-strength Hoagland nutrient solution. Plants were raised in a
greenhouse enhanced with high pressure sodium lamps (SON-T
AGRO, Philips, Belgium) at an intensity of 300 mmol m±2 s±1, 25±
30 °C, c. 80% relative humidity and a photoperiod of 16/8 h light/dark.
After 8 weeks, the seedlings were subjected to salt treatment. Salt
concentrations were stepped up in 50 mM d±1 increments until ®nal
concentrations (0, 100, 200, 300, 400 mM) were achieved. NaCl was
dissolved in Hoagland nutrient solution and plants were watered
daily to drip with approximate 0.5 l of salt solution. All measurements on the youngest and expanded leaves were made 3 weeks after
®nal treatment concentrations were reached, when plants had
achieved a steady state.
Heat stress was applied on detached leaves. The leaves were
placed directly into the smooth bottom of a small hole (5.5 cm in
height33 cm in diameter) in a block of brass (8 cm in height38 cm
in diameter), whose temperature was regulated by circulation of
water from a thermostated water bath. At the same time, the upper
surface of the leaves were directly pressed by a block of glass so that
the water evaporation of the leaf could be prevented and the heat
equilibrium between the leaves and the brass block could be reached
immediately. The hole in the brass was also covered during
treatment. The leaves were exposed to different elevated temperatures in the dark for 15 min and their ¯uorescence characteristics
were measured after heated leaves were kept at 25 °C for 5 min. No
difference was observed in the rate of temperature increase between
control and salt-adapted leaves. In addition, no changes were
observed in the various ¯uorescence parameters during the measurements of ¯uorescence after dark-adaptation of heat-stressed leaves
Measurements of chlorophyll ¯uorescence
Chlorophyll ¯uorescence was measured at room temperature with a
portable ¯uorometer (PAM-2000, Walz, Germany). The ¯uorometer
was connected to a computer with data acquisition software (DA2000, Walz). The experimental protocol of Genty et al. (1989) was
basically followed.
The minimal ¯uorescence level (Fo) with all PSII reaction centres
open was measured by the measuring modulated light, which was
suf®ciently low (<0.1 mmol m±2 s±1) not to induce any signi®cant
variable ¯uorescence. The maximal ¯uorescence level (Fm) with all
PSII reaction centres closed was determined by a 0.8 s saturating
pulse at 8000 mmol m±2 s±1 in dark-adapted leaves. The leaves were
then continuously illuminated with white actinic light at an intensity
PSII thermostability in salt-adapted plants 853
±2 ±1
of 300 mmol m s . The steady-state value of ¯uorescence (Fs) was
thereafter recorded and a second saturating pulse at 8000 mmol m±2
s±1 was imposed to determine the maximal ¯uorescence level in
light-adapted leaves (Fm¢). The actinic light was removed and the
minimal ¯uorescence level in the light-adapted state (Fo¢) was
determined by illuminating the leaf disc with 3 s far-red light. All
measurements of Fo and Fo¢ were performed with the measuring
beam set to a frequency of 600 Hz, whereas all measurements of Fm
and Fm¢ were performed with the measuring beam automatically
switching to 20 kHz during the saturating ¯ash.
By using ¯uorescence parameters determined in both light- and
dark-adapted leaves, calculations were made of: (1) the maximal
quantum yield of PSII photochemistry, Fv/Fm, (2) the photochemical
quenching coef®cient, qP=(Fm¢±Fs)/(Fm¢±Fo¢) and the non-photochemical quenching coef®cient qN=1±(Fm¢±Fo¢)/(Fm±Fo), (3) the
ef®ciency of excitation capture by open PSII reaction centres, Fv¢/
Fm¢, and (4) the actual PSII ef®ciency, FPSII. Fluorescence
nomenclature was according to van Kooten and Snel (1990).
The rapid ¯uorescence induction kinetics (Kautsky curve) were
measured in dark-adapted leaves suddenly illuminated with moderate white light of 45 mmol m±2 s±1 at a sampling rate of 1000 ms
point±1. In order to avoid an incomplete reoxidation of the
plastoquinone pool in the dark, which could result in an increase
in ¯uorescence level at phase I, the dark-adapted leaves were
illuminated with 3 s far-red light prior to measurement of the
¯uorescence induction kinetics.
The polyphasic rise of ¯uorescence transients (OJIP) was
measured by a Plant Ef®ciency Analyser (PEA, Hansatech
Instruments Ltd., King's Lynn, Norfolk PE32 1JL, UK) according
to Strasser et al. (1995). The transients were induced by red light of
3000 mmol m±2 s±1 provided by an array of six light-emitting diodes
(peak 650 nm), which focused on the sample surface to give
homogenous illumination over the exposed area of the sample (4 mm
in diameter). The ¯uorescence signals were recorded within a time
scan from 10 ms to 1 s with a data acquisition rate of 100 000 points
per second for the ®rst 2 ms and of 1000 points per second after 2 ms.
The ¯uorescence signal at 40 ms was considered as a true Fo since the
¯uorescence yield at this time was shown to be independent of light
intensity.
All samples were dark-adapted for 30 min prior to ¯uorescence
measurements.
Analysis of photosynthetic gas exchange
Gas exchange analysis was made using an open system (Ciras-1, PP
system, Hitchin, UK). Leaf net CO2 assimilation rate and stomatal
conductance (Gs) were determined at a CO2 concentration of
360 mmol mol±1, 80% relative humidity, and light intensity
(800 mmol m±2 s±1).
Measurements of the relative growth rate, leaf water
potential, and leaf water content
Relative growth rate was expressed as (Wt±Wo)/Wo, where Wo is the
fresh weight just before salt treatment, Wt is the fresh weight after 3
weeks salt treatment. Leaf water potential was measured with a
pressure chamber (Model 3000, Soil Moisture Equipment Co. USA)
on main branch shoots. Leaf water content was calculated as (FW±
DW)/FW, where FW is leaf fresh weight and DW is leaf dry weight.
Results
Relative growth rate, water potential, water content,
sodium and chloride contents, and CO2 assimilation
The growth of S. salsa was signi®cantly increased by salt
treatment. The relative growth rate increased signi®cantly
Fig. 1. (A) Relative growth rate, (B) water potential, (C) leaf water
content, and (D) sodium (®lled circles) and chloride (open inverted
triangles) contents of Suaeda salsa plants treated with various salt
concentrations for 3 weeks. Values represent mean 6SE of ®ve
replicates. Error bars are not shown if smaller than symbols.
with the increase in salt concentration ranging from 100 to
200 mM NaCl, but decreased slightly at 400 mM NaCl
(Fig. 1A), suggesting that 200±300 mM NaCl is optimal
for growth of S. salsa plants. With the increase of salt
concentration, leaf water potential declined considerably
(Fig. 1B). However, salinity showed no effects on water
content of leaves (Fig. 1C). Leaf sodium and chloride
contents in the salt-adapted plants increased dramatically
with the amount of salt added (Fig. 1D). The decrease in
leaf water potential without changes in water content was
854 Lu et al.
Fig. 2. Changes in CO2 assimilation rate (A) and stomatal
conductance (B) in Suaeda salsa plants treated with various salt
concentrations for 3 weeks. Values represent mean 6SE of 3±4
replicates. Error bars are not shown if smaller than symbols.
attributed mainly to the accumulation of sodium and
chloride (Qiu et al., 2001).
Figure 2 shows the changes in net CO2 assimilation rate
and stomatal conductance in salt-adapted plants. CO2
assimilation rate increased slightly with increasing salt
concentration up to 300 mM NaCl and showed no decrease
even at 400 mM NaCl, whereas stomatal conductance
decreased slightly with increasing in salt concentration.
PSII photochemistry in salt-adapted leaves
The changes in PSII photochemistry were ®rst investigated
in dark-adapted state in salt-adapted leaves. Figure 3A
shows that no changes in the maximal ef®ciency of PSII
Fig. 3. (A) The maximum ef®ciency of PSII photochemistry (Fv/Fm),
(B) the proportion of QB-non-reducing PSII reaction centres,
expressed as (Fi±Fo)/(Fp±Fo), in Suaeda salsa plants treated with
various salt concentrations for 3 weeks. (Inset) the rapid ¯uorescence
induction kinetics (O-I-P) in dark-adapted control leaves suddenly
illuminated with moderate white light (40 mmol m±2 s±1). Each value is
mean 6SE of four replicates. Error bars are not shown if smaller than
symbols.
photochemistry (Fv/Fm) measured in dark-adapted leaves
were observed in salt-adapted plants.
In order to examine whether salinity treatment induces
the changes in the heterogeneity of PSII reaction centres,
the rapid ¯uorescence induction kinetics was thus determined (Klinkovsky and Naus, 1994). The inset in Fig. 3B
shows the rapid ¯uorescence induction kinetics of S. salsa
leaves, which consists of three phases, i.e. O, I and P. The
ratio (Fi±Fo)/(Fp±Fo) is a measure of the percentage of
those QB-non-reducing PSII reaction centres (Chylla and
Whitmarsh, 1989; Cao and Govindjee, 1990). Figure 3B
PSII thermostability in salt-adapted plants 855
Fig. 4. The polyphasic chlorophyll ¯uorescence transients plotted on a
logarithmic time scale in Suaeda salsa plants treated with various salt
concentrations for 3 weeks.
shows that no changes in this ratio were observed in saltadapted plants, indicating that salinity treatment had no
effect on the proportion of QB-non-reducing PSII reaction
centres.
It has been demonstrated that when a dark-adapted leaf
is illuminated with high intensity actinic light (3000 mmol
m±2 s±1), the ¯uorescence transient shows a polyphasic rise,
including phases O, J, I, and P (Strasser et al., 1995). The
initial Chl ¯uorescence at O level re¯ects the minimal
¯uorescence yield when all molecules of QA are in the
oxidized state. The P level corresponds to the state in
which all molecules of QA are in the reduced state. Steps J
and I occur between the commonly observed steps O and
P. The rise from phase O to phase J results from the
reduction of QA to QA± and is associated with the primary
photochemical reactions of PSII. The intermediate step I
and the ®nal step P re¯ect the existence of fast and slow
reducing PQ centres, as well as to the different redox states
of PSII reaction centres (Strasser et al., 1995). Thus,
compared to the traditional Kautsky curve, the polyphasic
rise of ¯uorescence transients can give more information
on PSII photochemistry.
Therefore, the polyphasic Chl a ¯uorescence transients
in salt-adapted leaves was followed. The polyphasic rise in
Chl a ¯uorescence transients in S. salsa leaves demonstrated a typical polyphasic rise, including phases O, J, I,
and P, described in detail by Strasser et al. (1995). Salinity
treatment had no signi®cant effect on these phases in the
polyphasic rise of ¯uorescence transients (Fig. 4).
Fig. 5. (A) The actual PSII ef®ciency (FPSII, ®lled circles) and the
ef®ciency of excitation capture by open PSII reaction centres (Fv¢/Fm¢,
open inverted triangles), (B) the photochemical quenching coef®cient
(qP, ®lled circles) and the non-photochemical quenching coef®cient
(qN, open inverted triangles) in Suaeda salsa plants treated with
various salt concentrations for 3 weeks. Values represent mean 6SE
of ®ve replicates. Error bars are not shown if smaller than symbols.
The above results show that no effects on the primary
photochemistry of PSII determined in dark-adapted leaves
were observed in salt-adapted plants. It was investigated
further whether salinity treatment induced modi®cations in
PSII photochemistry in light-adapted leaves. the changes
in ¯uorescence characteristics in light-adapted leaves were
examined by use of pulse-modulated ¯uorescence.
Figure 5 shows that salinity treatment showed no effects
on the ef®ciency of excitation energy capture by open PSII
reaction centres (Fv¢/Fm¢), the actual PSII ef®ciency
(FPSII), the photochemical quenching coef®cient (qP), as
well as the non-photochemical quenching coef®cient (qN).
856 Lu et al.
Fig. 6. CO2 assimilation rates in control (0 mM NaCl) and saltadapted (100±400 mM NaCl) leaves of Suaeda salsa plants exposed to
elevated temperatures in the dark for 15 min. The values in the ®gure
are expressed as percentages of the values of CO2 assimilation rates at
30 °C which are shown in Fig. 2. Values represent mean 6SE of 3±4
replicates. Error bars are not shown if smaller than symbols.
Thermostability of CO2 assimilation and PSII
photochemistry in salt-adapted leaves
The changes in CO2 assimilation rate in salt-adapted leaves
were examined after exposure to high temperatures
(Fig. 6). CO2 assimilation rate decreased signi®cantly
with increasing temperature. No net CO2 assimilation was
observed at all at 47.5 °C. Salinity treatment had no effect
on CO2 assimilation at temperatures below 40 °C. When
the temperature was at 45 °C, the CO2 assimilation rate in
control plants decreased to zero, but the salt-adapted plants
still maintained some CO2 assimilation capacity, which
was about 15±20% higher than control plants.
The effects of salinity treatment on the thermostability
of PSII were investigated as well as the effects of the
degree of salinity on the thermostability of PSII. No
signi®cant difference was observed in the responses of
PSII photochemistry (all of the parameters in dark- and
light-adapted leaves) to high temperature in S. salsa plants
treated with different NaCl concentrations, suggesting that
the increased thermostability of PSII induced by salinity
treatment, as shown below, is independent of the degree of
salinity. Here, only the results for S. salsa plants treated
with 400 mM NaCl are presented.
The responses of PSII photochemistry in the darkadapted state to high temperatures were investigated ®rst.
Figure 7A shows that salt-adapted plants exhibited much
higher values of Fv/Fm than the control plants under high
Fig. 7. (A) The maximum ef®ciency of PSII photochemistry (Fv/Fm),
(B) the actual PSII ef®ciency (FPSII), (C) the ef®ciency of excitation
capture by open PSII reaction centres (Fv¢/Fm¢), and (D) the
photochemical quenching coef®cient (qP) in control 0 mM NaCl, ®lled
circles) and salt-adapted (400 mM NaCl, open inverted triangles)
leaves of Suaeda salsa plants exposed to elevated temperatures in the
dark for 15 min. Each value is mean 6SE of four replicates. Error
bars are not shown if smaller than symbols.
temperatures. For example, at 47.5 °C, Fv/Fm decreased by
53% in control plants, but only by 25% in salt-adapted
plants.
The responses of PSII photochemistry in light-adapted
leaves, i.e. under steady-state photosynthesis, to high
temperatures were examined further. Figure 7B±D shows
that a greater decrease in FPSII Fv¢/Fm¢, and qP was
observed in control plants than in salt-adapted plants in the
PSII thermostability in salt-adapted plants 857
temperature range 35±47.5 °C. The results from Fig. 6
indicate that salinity treatment induced an increase in the
resistance of PSII to heat stress.
It has been shown that the O2-evolving complex (OEC)
is the most susceptible component in the PSII apparatus to
heat stress (Berry and BjoÈrkman, 1980), therefore whether
the increased thermostability of PSII induced by salinity
treatment was associated with an improvement in the
thermostability of the OEC was examined.
As shown in Fig. 8A, both control and salt-adapted
plants show a typical polyphasic rise of ¯uorescence
transients, including phases O, J, I, and P. It has been
shown that heat stress can induce a rapid rise in the
polyphasic ¯uorescence transients. This phase, occurring
at around 300 ms, has been labelled as K, and is the fastest
phase observed in the OJIP transient which, in consequence, becomes an OKJIP transient (Guisse et al., 1995;
Srivastava and Strasser, 1996; Srivastava et al., 1997). It
has also been shown that phase K is caused by an inhibition
of the electron donor to the secondary electron donor of
PSII, YZ, which is due to a damaged OEC, amplitude of the
step K can therefore be used as a speci®c indicator of
damage to the OEC (Guisse et al., 1995; Strasser, 1997).
Figure 8A shows that when exposed to 47.5 °C for
15 min, a very clear K step appeared in control plants.
However, this heat-induced K step was not pronounced in
salt-adapted plants. The changes in the amplitude in the K
step, expressed as the ratio VK, was shown in Fig. 8B. A
higher ratio VK was observed in control plants than in saltadapted plants from 35±47.5 °C, indicating that salinity
treatment can increase the resistance of the OEC to heat
stress.
It has been suggested that the PSII reaction centre is one
of the damage sites induced by heat stress (Klinkovsky and
Naus, 1994; Cao and Govindjee, 1990). It was therefore
investigated whether increased thermostability of PSII
induced by salinity treatment was associated with an
increase in the resistance of the PSII reaction centres to
heat stress. It has been shown that heat stress induced an
increase in the amplitude of the O-I phase in the Kautsky
curve and (Fi±Fo)/(Fp±Fo), an expression of the proportion
of the QB-non-reducing PSII centres, has been shown to be
sensitive to heat stress (Klinkovsky and Naus, 1994).
similar results were observed when heat stress induced an
increase in the amplitude of the O-I phase and thus an
increase in the ratio (Fi±Fo)/(Fp±Fo), suggesting that heat
stress resulted in an inactivation of PSII reaction centres.
The insert of Fig. 9 gives an example of the changes in the
amplitude of the O-I phase after the control leaves were
exposed to 47.5 °C. Moreover, a greater increase in the
ratio (Fi±Fo)/(Fp±Fo) was observed in control plants than
in salt-adapted plants, suggesting that salinity treatment
also induced an increase in the resistance of PSII reaction
centres to heat stress (Fig. 9).
Fig. 8. (A) Effects of high temperature on the polyphasic chlorophyll
¯uorescence transients plotted on a logarithmic time scale in control
and salt-adapted leaves of Suaeda salsa. The curves are: (open circles)
control leaves, (®lled circles) salt-adapted leaves, (open inverted
triangles) control leaves exposed to 45 °C for 15 min, and (®lled
inverted triangles) salt-adapted leaves exposed to 45 °C. The relative
variable ¯uorescence at any time t is de®ned as: Vt=(Ft±Fo)/(Fm±Fo).
(B) Changes in the ratio VK, expressed (FK±FO)/(FP±FO), in control
(0 mM NaCl, ®lled circles) and salt-adapted (400 mM NaCl, open
inverted triangles) leaves after exposed to elevated temperatures in the
dark for 15 min. Each value is mean 6SE of four replicates. Error
bars are not shown if smaller than symbols.
Discussion
In the present study, it was observed that PSII in S. salsa
showed a high resistance to high salinity. This is re¯ected
858 Lu et al.
Fig. 9. Changes in the proportion of QB-non-reducing PSII reaction
centres expressed as (Fi±Fo)/(Fp±Fo) in control (0 mM NaCl, ®lled
circles) and salt-adapted (400 mM NaCl, open inverted triangles)
leaves of Suaeda salsa exposed to elevated temperatures in the dark
for 15 min. Each value is mean 6SE of four replicates. Error bars are
not shown if smaller than symbols. The inset: the rapid ¯uorescence
induction kinetics curves (O-I-P) in dark-adapted leaves suddenly
illuminated with moderate white light (40 mmol m±2 s±1). Curves (a)
and (b) are control leaves exposed to 30 °C and 47.5 °C, respectively,
in the dark for 15 min.
by the fact that no signi®cant changes were observed in the
¯uorescence parameters related to PSII photochemistry
either in the dark-adapted state (Figs 3, 4) or in the lightadapted state (Fig. 5) when S. salsa plants were treated
with NaCl as high as 400 mM. These results suggest that
S. salsa, a halophyte plant, is capable of adapting to high
salinity.
An increase in the resistance of PSII to high temperatures had previously been observed after sorghum plants
were adapted to high salinity, suggesting that salinity
treatment can induce an increased thermostability of PSII
(Lu and Zhang, 1998). In general, sorghum is an agricultural crop that is sensitive to salinity stress. It is postulated
that halophyte plants which are capable of adapting to high
salinity also should have an ability to adapt to high
temperatures after they are adapted to high salinity. In this
study, a previous study was extended to investigate if
salinity treatment can also induce an increase in thermostability of PSII in S. salsa, a halophyte plant, after they are
adapted to high salinity. It was demonstrated that salinity
treatment also enhanced the resistance of PSII to heat
stress in S. salsa plants (Fig. 7). It is also shown that such
increased thermostability is independent of the degree of
salinity, since there was no signi®cant difference in the
responses of the PSII photochemistry to heat stress in
plants treated with different NaCl concentrations
(100±400 mM).
Since sorghum and S. salsa differ signi®cantly in growth
response to high salinity, the increased thermostability of
PSII observed in both plants suggests that the protective
effects on PSII functioning, induced by salinity treatment,
may be not associated with whole plants and growth
effects. The comparison between salt-sensitive plants
(sorghum) and halophytes (S. salsa) may provide physiological evidence for the suggestion that the two types of
plants have homology with respect to molecular responses
to salinity stress (Zhu, 2001). Moreover, it is interesting
that, similar to salinity treatment, drought stress also leads
to an increase in the thermostabilty of PSII (Lu and Zhang,
1999), suggesting that salinity treatment and water stress
may have common mechanisms that induce an increase in
the resistance of PSII to heat stress. Thus, these results
suggest that the increased thermostability induced by
salinity treatment on PSII in sorghum and S. salsa may be
part of a general response to osmotic stress. Indeed, plants
genetically engineered to be salt tolerant are often shown
to be tolerant to a variety of other stresses, such as drought
stress and low temperature.
Since the O2-evolving system has shown to be most
sensitive to heat stress (Berry and BjoÈrkman, 1980), it was
investigated if an increase in thermostability of PSII in
salt-adapted S. salsa plants was associated with an increase
in thermostability of the O2-evolving complex (OEC). The
results demonstrated that the increased thermostability of
PSII induced by salinity treatment was related to an
increase in thermostability of the OEC, as shown by an
appearance of phase K in the polyphasic ¯uorescence
transients in control plants (Fig. 8). In addition, the PSII
reaction centre is one of the damage sites induced by heat
stress (Klinkovsky and Naus, 1994; Cao and Govindjee,
1990). The results show that the increased thermostability
of PSII induced by salinity treatment was partly associated
with an increase in the thermostability of the PSII reaction
centre. This is re¯ected in the increased thermostability of
PSII reaction centres, since it was observed that high
temperature induced a smaller increase in the proportion of
QB-non-reducing PSII reaction centres in salt-adapted
plants than in control plants (Fig. 9). This is also con®rmed
by a smaller decrease in qP in salt-adapted plants (Fig. 7D),
which can represent the fraction of open PSII reaction
centres (Genty et al., 1989; van Kooten and Snel, 1990).
The results of this study seem to support the hypothesis
that an increased resistance of PSII to heat stress in saltadapted leaves was involved in several aspects of PSII
functioning, such as the O2-evolving complex and the PSII
reaction centre.
How salinity treatment can protect PSII against heat
damage is not clear. It has been shown that the resistance
of the thylakoids to heat stress is enhanced when the
chloroplasts are incubated in the presence of some soluble
PSII thermostability in salt-adapted plants 859
compounds, such as sugars and proteins (Krause and
Santarius, 1975; Santarius, 1975). It was indeed found that
the accumulation of these soluble compounds existed in
salt-stressed leaves (Greenway and Munns, 1980; Imamul
Huq and Larher, 1984). In addition, the accumulation of
these soluble compounds also existed in water-stressed
leaves (Seemann et al., 1986; Sachs and Ho, 1986).
Moreover, it has been shown that the osmolytes, such as
glycinebetaine, can stabilize the oxygen-evolving complex
and protect the oxygen-evolving complex, together with
the PSII core, from heat stress (Murata et al., 1992;
Papageorgiou and Murata, 1995; Allakhverdiev et al.,
1996). Glycinebetaine is synthesized in the chloroplast and
can be accumulated in chloroplasts up to 1 M when some
plants are grown in saline environments. Thus, it is
possible that the increased thermostability of PSII in
S. salsa plants may be explained, at least partially, by the
accumulation of these protective compounds, such as
glycinebetaine, in salt-adapted leaves. The exact mechanisms for increased thermostability of PSII induced by
salinity treatment remain to be elucidated.
Photosynthesis has been shown to be the main site of
damage from high temperature and PSII within the
photosynthetic apparatus appears to be most heat sensitive
(Berry and BjoÈrkman, 1980). A rapid adaptation of PSII to
high temperature in salt-adapted leaves, with the increased
thermostability of PSII, is of great physiological signi®cance because such an increased resistance of PSII to high
temperature may help to improve the resistance of the
photosynthetic metabolism to heat stress and thus may
increase the resistance of the whole plant to heat stress. In
this sense, the increased thermostability of PSII to heat
stress in salt-adapted leaves can be seen as a very
important strategy for plants withstanding heat stress
during salt stress, particularly in arid and semi-arid regions
where salinity stress is often accompanied by high
temperature. It has to be further investigated whether
such an increased thermostability of PSII to heat stress
induced by salinity treatment can really contribute to the
overall ability of plants to survive long periods of salinity
stress.
Acknowledgements
This work was supported by the Program of 100 Distinguished
Young Scientists of the Chinese Academy of Sciences, the State Key
Basic Research and Development Plan of China (No. G1998(10100)
(to CL), as well as Hong Kong Research Grants Council (HKBU
2041/01M to JZ).
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