Botanical Studies (2006) 47: 61-69.
*
Corresponding author: E-mail: jianlongli@sina.com; Tel:
+86-25-86214644; Fax: +86-25-83302728.
High temperature effects on photosynthesis, PSII
functionality and antioxidant activity of two
Festuca
arundinacea
cultivars with different heat susceptibility
Langjun CUI, Jianlong LI*, Yamin FAN, Sheng XU, and Zhen ZHANG
School of Life Science, Nanjing University, Hankou Road 22, Nanjing 210093, P.R. China
(Received June 27, 2005; Accepted October 7, 2005)
ABSTRACT.
High temperature stress is a major limiting factor in the growth and development of tall
fescue (Festuca arundinacea) in transitional and warm climatic regions. In this study, we evaluated the
photosynthesis, PSII functionality, and antioxidant activity of two tall fescue cultivars, Jaguar 3 brand
(heat-tolerant) and TF 66 (heat-sensitive) in response to high temperature stress. High temperature stress
caused a net photosynthetic rate reduction in the two plants due to stomatal and non-stomatal limitations,
photoinhibition increase, and Rubisco activity reduction. High temperature stress modified PSII functionality
in leaves of the two plants, manifested by lower variable chlorophyll fluorescence yield (Fv), maximum
photochemical efficiency of photosystem .. in dark adapted leaves (Fv/Fm), and efficiency of the open
reaction centre in light (ΦPSII
open
) in the two heat shocked cultivars. Heat stress led to reductions in the
chlorophyll a+b and chlorophyll/carotenoid ratios and to an increase in the chlorophyll a/b ratio for the two
stressed cultivars. Moreover, high temperature stress significantly increased lipid peroxidation, decreased
cell membrane thermostability, and changed the activities of ascorbate peroxidase (APX) and superoxide
dismutase (SOD) in the leaves of both plants. All the above effects induced by high temperature were more
expressed in the TF 66 than in Jaguar 3 brand. Our results contain some insights which may prove useful in
the selection and breeding of heat-tolerant tall fescue turfgrass cultivars.
Keywords: Antioxidant enzyme; Cell membrane integrity; Festuca arundinacea; High temperature stress;
Photosynthetic characteristics; PSI. photochemical efficiency.
Abbreviations: A
max-CO2
, maximum net photosynthesis under maximum Ci; A
max-light
, maximum net photo-
synthesis at maximum PPFD level; AOS, active oxygen species; APX, ascorbate peroxidase; A
sat-CO2
, CO
2
-
saturated net photosynthetic rate; A
sat-light
, light saturated net photosynthetic rate; CE, carboxylation efficiency;
Chl, chlorophyll; Ci, internal CO
2
concentration; E, transpiration rate; EL, electrolyte leakage; Fv, variable
chlorophyll fluorescence yield; Fv/Fm, maximum photochemical efficiency of photosystem .. in dark adapted
leaves; g
s
, stomatal conductance; LCP, light compensation point; LSP, light saturating point; MDA, malondi-
aldehyde; Pn, net photosynthetic rate; PPFD, photosynthetic photo flux density; PSII, photosystem II; Rd,
light respiration; SOD, superoxide dismutase; WUE, water use efficiency; Γ, CO
2
compensation point; Φ, ap-
parent quantum efficiency; ΦPSII
open
, efficiency of the open reaction centre in light.
INTRODUCTION
High temperature stress is one of the major factors
limiting use of cool-season grasses in transitional and
warm climatic regions (Carrow, 1996; Beard, 1997). High
temperature for prolonged periods during midsummer in
these regions can inhibit cool-season grass growth (Martin
and Wehner, 1987), decrease turf quality, and reduce
photosynthetic rate (Huang and Gao, 2000).
Photosynthesis, one of the most heat sensitive
processes, can be completely inhibited by high
temperature before other symptoms of the stress are
detected (Camejo et al., 2005). This photosynthesis
decrease could result from structural and functional
disruptions of chloroplasts and reduction of chlorophyll
accumulation under high temperature stress (Xu et al.,
1995; Dekov et al., 2000). Moreover, Higuchia et al.
(1999) reported that photosynthesis showed heat inhibition
of both stomatal limitation and non-stomatal limitation in
cherimoya while Camejo et al. (2005) suggested that there
was no stomatal limitation in tomato.
PHYSIOLOGY
pg_0002
62
Botanical Studies, Vol. 47, 2006
The reduction of photosynthesis by high temperature
stress is also related to inactivation of many chloroplast
enzymes, mainly induced by oxidative stress (Dekov
et al., 2000). Oxidative stress can cause cause lipid
peroxidation and consequently membrane injury, protein
degradation, and enzyme inactivation (Sairam et al., 2000;
Meriga et al., 2004). Therefore, the ability to maintain cell
membrane integrity and diminish oxidative stress have
been proposed as good indicators of thermotolerance in
plants (Liu and Huang, 2000; Huang et al., 2001).
Tall fescue (Festuca arundinacea) is one of the most
widely used cool-season species on golf putting greens.
The optimum temperatures for tall fescue cultivation are
between 15 and 20°C. However, temperatures of 30 to
35°C in the transitional and warm climatic regions dur-
ing midsummer are common and can heavily reduce turf
quality and cultivar growth. Therefore, this work aims to
determine how two tall fescue grasses that differ in their
tolerance to high temperature stress respond to it in their
photosynthesis, PSII functionality and antioxidant ability
to diminish oxidative stress. Based on these comparisons,
insight into the mechanism responsible for differences in
thermotolerance for tall fescue cultivars should be pos-
sible.
Materials and Methods
Plant materials and treatments
Seeds of two tall fescue (Festuca arundinacea S.)
cultivars, Jaguar 3 brand (heat tolerant), and TF 66 (heat
sensitive) were sown and grown in a mixture of sand,
vermiculite, and organic matter (3:1:1) in polyethylene
pots (14 cm in height and 13 cm in diameter with 175
seeds in each pot). Each grass was planted in twelve pots.
Plants were kept in growth chambers at 20/15°C (day/
night), a photosynthetic photo flux density (PPFD) of 200
μmol m
-2
s
-1
, and a 14-h photoperiod for 70 d (including
8 d for germination) before they were subjected to high
temperature stress. Then six pots of each grass were trans-
ferred to growth chambers with high temperature 35/30
°C (day/night) for 20 d, with the remaining pots kept un-
der control conditions 20/15°C (day/night). Before and
during the temperature treatments, grasses were mowed
weekly to a height of 8 cm with electric hair clippers, wa-
tered daily, and fertilized weekly with a full Hoagland’s
nutrient solution (Hoagland and Arnon, 1950).
Gas-exchange measurements
Gas-exchange measurements were taken on healthy
leaves using a portable Licor 6400 photosynthesis system
(LI-6400, Li-Cor Inc., Lincoln NE, USA). The measure-
ments were conducted under 20 or 35°C with 400 μmol
CO
2
mol
-1
air CO
2
concentration and 1000 μmol m
-2
s
-1
PPFD (supplied by a red-blue LED light source). Photo-
synthetic water use efficiency (WUE) was determined by
the ratio of net photosynthetic rate (Pn) to transpiration
rate (E).
Photosynthetic irradiance and CO
2
response
Measurements of leaf photosynthetic irradiance and
CO
2
response curves were made using a Licor 6400
photosynthesis system (LI-6400, Li-Cor Inc., Lincoln
NE, USA). Artificial illumination was supplied to the
leaf from a red-blue LED light source, and ambient CO
2
partial pressure was supplied by 6400 CO
2
mixer. A pho-
tosynthetic irradiance response curve (A-PPFD curve)
was monitored using the following procedures: healthy
leaf was illuminated at a PPFD of 800 μmol m
-2
s
-1
un-
til a steady state of net CO
2
fixation was reached at 400
μmol CO
2
mol
-1
air CO
2
concentration. Irradiance was
then changed in a stepwise reduction of PPFD ranging
from 1800 to 0 μmol m
-2
s
-1
and measurements were made
once the leaf attained a steady net CO
2
fixation rate. The
asymptotic exponential equation of Prioul and Chartier
(1977) was fitted by non-linear least square regression to
the A-PPFD data. Fixed parameters of the model were es-
timated using a SPSS 10.0 statistical package.
Photosynthetic response to different internal CO
2
concentrations (Ci) (A-Ci curve) was measured for the
photosynthetic irradiance response curves with the ex-
ception that once steady state was attained, ambient CO
2
partial pressure in the leaf chamber (Ca) was reduced
in steps from 400 μmol CO
2
mol
-1
air down to 0 μmol
CO
2
mol
-1
air, and then increased up to 1400 μmol CO
2
mol
-1
air, with a saturated PPFD 1200 μmol m
-2
s
-1
. Net
photosynthetic rate (Pn) of the leaves of the two grasses
was measured at 20 or 35°C. A-Ci parameters were calcu-
lated by the model of Olsson and Leverenz (1994).
Pigment content determination
The content of chlorophyll (Chl) a, b and carotenoid
in leaf segments was determined in 80% acetone follow-
ing Lichtenthaler’s (1987) method using a UV-VISIBLE
Spectrophotometer (Hitachi U-3000).
Leaf photochemical efficiency measurements
Leaf photochemical efficiency was estimated with
a plant photosynthesis efficiency analyzer (Hansatech
Instrument LTD, Kings Lynn, England). Initial chlorophyll
fluorescence yield, Fo, variable chlorophyll fluorescence
yield, Fv, and maximum chlorophyll fluorescence yield,
Fm, were read in the fluorometer. Attached leaves were
covered in a leaf chamber and adapted in the dark for 20
min before measurements were conducted. Maximum
photochemical efficiency of the photosystem .. in dark
adapted leaves (Fv/Fm) and efficiency of the open reaction
centre in light (ΦPSII
open
) were determined using follow-
ing equations:
Fv/Fm=(Fm-Fo)/Fm
ΦPSII
open
=Fv’/Fm’=(Fm’-Fo)/Fm’
Lipid peroxidation and membrane thermostability
The level of lipid peroxidation in leaf and root tissue
was measured in terms of malondialdehyde (MDA) con-
pg_0003
CUI et al. — High temperature on tall fescue
63
tent, which was determined by the thiobarbituric acid re-
action following the procedure of .pundova et al. (2003).
Membrane thermostability was determined by measuring
electrolyte leakage (EL). EL was measured by conductiv-
ity method according to Gulen and Eris (2004).
Enzyme extraction and enzyme assays
A 0.2 g of leaf tissue was harvested into liquid nitrogen
at various times throughout the study and stored at -80
°C. On removal from the -80°C freezer, the samples were
immediately placed in liquid nitrogen, removed, and
immediately ground with a micropestle in an eppendorf
in 0.25 mL of cold phosphate buffer (50 mmol/L, pH
7.0) containing 0.2 mmol/L ascorbate and 1% polyvinyl-
polypyrrolidone. The ground tissue was then spun at 0°C
for 15 min in a microfuge. The resulting extract was stored
on ice for as little time as possible prior to taking readings.
All of the enzyme assays were measured by the pro-
cedures of Larkindale and Huang (2004). The assay for
ascorbate peroxidase (APX) (EC 1.11.1.11) was done by
monitoring the rate of oxidation of ascorbate at 290 nm
using the spectrophotometer blanked against an aliquot of
buffer (50 mmol/L phosphate buffer, pH 7.0, 0.5 mmol/L
ascorbate, 0.1 mmol/L EDTA).
Superoxide dismutase (SOD) (EC 1.15.1.1) was mea-
sured at 560 nm, and 1 unit of activity was defined as the
amount of enzyme required to inhibit 50% of the NBT
reduction rate in the controls containing no enzyme.
All samples were corrected for the amount of total pro-
tein in the extract, which was measured according to the
method of Bradford (1976).
Statistical analysis
One-way analysis of variance was performed using the
SPSS computer package (SPSS Inc. 1999) for all sets of
data, and means were compared using Duncan’s multiple
comparison test at P = 0.05.
RESULTS
The effect of high temperature on photosynthetic
rate and WUE
Net photosynthetic rate (Pn) and stomatal conductance
(g
s
) in the two treated cultivars were reduced after high
temperature stress compared to controls (Figure 1A, B).
Transpiration rate (E) in both plants increased under heat
stress (Figure 1C). Under this stress condition, no signifi-
cant variations in the internal CO
2
concentrations (Ci) for
the two cultivars were measured (Figure 1D). Water use
efficiency (WUE) in both treated cultivars fell after high
temperature stress compared to controls (Figure 2). Pn
in Jaguar 3 brand was always higher than in TF 66. The
difference in the g
s
between
the control plants of Jaguar 3
brand and TF 66 was not remarkable while the g
s
in Jaguar
3 was higher than in TF 66 after 10 d stressed treatment
and then lower after 20 d. Compared to TF 66, Jaguar 3
had lower E under control conditions and a higher E under
high temperature stress. Under high temperature stress,
the rates of increase in E for Jaguar 3 were significantly
higher than those in TF 66. WUE in Jaguar 3 brand was
higher than in TF 66 under control conditions, but lower
under stressed conditions.
A-PPFD curves parameters
Analyses of A-PPFD curves showed that both appar-
ent quantum efficiency (Φ), maximum net photosynthesis
(A
max-light
) at maximum PPFD, and light saturated net pho-
tosynthetic rate (A
sat-light
) (PPFD = 800 μmol m
-2
s
-1
) for
the two stressed cultivars were decreased when compared
to each control (Figure 3, Table 1). The light compensa-
Figure 1. Net photosynthetic rate, Pn (A), stomatal conduct-
ance, g
s
(B), transpiration rate, E (C) and internal CO
2
concentra-
tions Ci, (D) in leaves of two tall fescue (Festuca arundinacea)
cultivars (Jaguar 3 brand and TF 66) grown under normal and
high temperature conditions. Measurements were conducted at
20 or 35°C. Each point is the mean ± S.E. of 3-6 leaves.
Figu re 2. Water use efficiency (WUE) in leaves of two tall
fescue (Festuca arundinacea) cultivars (Jaguar 3 brand and TF
66) grown under normal and high temperature conditions. Mea-
surements were conducted at 20 or 35°C. Each point is the mean
± S.E. of 3-6 leaves.
pg_0004
64
Botanical Studies, Vol. 47, 2006
tion point (LCP) of the two cultivars decreased after 10 d
treatment and then increased after 20 d treatment. In com-
parison to control plants, the light saturation point (LSP)
of the two heat stressed cultivars increased (Table 1).
Jaguar 3 brand had higher A
max-light
, A
sat-light
and Φ than TF
66, whether treated or not. Differences of LCP and LSP
between Jaguar 3 brand and TF 66 were not significant
under normal or stressed conditions (Figure 3, Table 1).
A-Ci curves parameters
The curves, which characterize Pn, vs. Ci, were pre-
sented in Figure 4. Within the range of 12-560 μmol CO
2
mol
-1
air Ci, essentially a single-line dependence of Pn
was observed. The further increase of Ci concentration
only slightly influenced the rate of Pn. Both maximum net
photosynthesis under maximum Ci (A
max-CO2
) and CO
2
-
saturated Pn (A
sat-CO2
) (Ca = 400 μmol CO
2
mol
-1
air) for
the two cultivars were reduced by high temperature stress
compared to the control plants (Figure 4, Table 2). Heat
stress increased light respiration (Rd) and CO
2
compen-
sation point (Γ) in the two treated plants. Compared to
the control, carboxylation efficiencies (CEs) of the heat
stressed plants were lower except for a slight increase
for Jaguar 3 brand at 10 d heat stress (P<0.05) (Table 2).
Jaguar 3 brand had higher A
max-CO2
, A
sat-CO2
and CE than TF
66 under normal and stressed conditions while a reverse
phenomenon was found for Rd and Γ, but the differences
in A
sat-CO2
and CE were not reliable.
Figure 3. The relationship between net photosynthetic rate
(Pn) and photosynthetic photon flux density (PPFD) in leaves of
two tall fescue (Festuca arundinacea) cultivars (Jaguar 3 brand
and TF 66) grown under normal and high temperature condi-
tions. Measurements were conducted at 20 or 35°C, with a CO
2
concentration around 400 μmol CO
2
mol
-1
air. Each point is the
mean ± S.E. of 3-6 leaves.
Table 1. Average parameters of A-PPFD curves for two tall fescue (Festuca arundinacea) cultivars (Jaguar 3 brand and TF 66)
grown under normal and high temperature conditions.
Jaguar 3 brand
TF 66
Control
10 d stress 20 d stress
Control
10 d stress 20 d stress
A
max-light
11.50±0.24
a
9.80±0.41
c
10.65±0.33
b
10.2±0.28
a
9.47±0.56
b
9.45±0.35
b
A
sat-light
10.16±0.45
a
9.19±0.30
b
8.24±0.19
c
9.71±0.73
a
8.07±0.35
b
5.87±0.45
c
.
0.080±0.003
a
0.048±0.009
c
0.063±0.005
b
0.061±0.003
a
0.044±0.001
c
0.054±0.006
b
LCP
7.35±0.52
b
6.72±0.55
b
13.68±1.16
a
8.72±0.75
b
7.16±0.63
c
12.9±2.59
a
LSP
303.1±10.2
c
372.1±20.2
a
330.3±18.6
b
284.3±11.7
b
324.7±20.2
a
358.0±18.1
a
These parameters include maximum net photosynthesis at maximum PPFD level (A
max-light
) (μmol CO
2
m
-1
s
-1
), light saturated net
photosynthetic rate (A
sat-light
) (μmol CO
2
m
-1
s
-1
), apparent quantum efficiency (Φ) (μmol CO
2
μmol
-1
photon), light compensation
point (LCP) (μmol m
-2
s
-1
) and light saturating point (LSP) (μmol m
-2
s
-1
). Each value is the mean ± S.E. based on six determina-
tions.
Figure 4. The relations hip between net photosynthetic rate
(Pn) and internal CO
2
concentrations (Ci) in leaves of two tall
fescue (Festuca arundinacea) cultivars (Jaguar 3 brand and
TF 66) grown under normal and high temperature conditions.
Measurements were conducted at 20 or 35°C, with a saturated
photosynthetic photon flux density (PPFD) 1200 μmol m
-2
s
-1
.
Each point is the mean ± S.E. of 3-6 leaves.
pg_0005
CUI et al. — High temperature on tall fescue
65
Chlorophyll and carotenoid content and leaf
photochemical efficiency
Heat Stress altered chlorophyll (Chl) and carotenoid
content and leaf photochemical efficiency in the two
stressed plants (Figure 5). Both Chl a+b content and Chl/
carotenoid ratio fell in the two heat stressed cultivars in re-
lation to the control plants (Figure 5A, C). An increase in
the Chl a/b ratio occurred in the two treated plants (Figure
5B), caused mainly by a higher decrease in Chl b content
than Chl a content (data not shown). Both variable chloro-
phyll fluorescence yield (Fv) and maximum photochemi-
cal efficiency of photosystem .. in dark adapted leaves
(Fv/Fm), as well as efficiency of the open reaction centre
in light (ΦPSII
open
), were reduced in the two stressed cul-
tivars at the end of heat shock (Figure 5D, E, F). Jaguar
3 brand retained a significantly higher Chl a+b content,
Chl/carotenoid ratio, and Fv and Fv/Fm ratio than did TF
66 during high temperature stress. The Chl a/b ratio in TF
66 was much higher than in Jaguar 3 brand after 10 d and
20 d treatment. Moreover, ΦPSII
open
in Jaguar 3 brand was
higher than in TF 66 at normal and stressed conditions,
but the difference was not significant at 20 d treatment
(P<0.05).
Lipid peroxidation, membrane thermostability,
and antioxidant enzyme activities
High temperature stress increased leaf membrane per-
oxidation and decreased membrane thermostability in the
two stressed cultivars. Malondialdehyde (MDA) content
and electrolyte leakage (EL) level increased significantly
in both stressed cultivars (Figure 6A, B). During high
temperature stress, TF 66 maintained a significantly higher
content of MDA and level of EL than Jaguar 3 brand did
(P<0.05).
Activity of ascorbate peroxidase (APX) in the two cul-
tivars increased after 10 d heat stress while it decreased re-
markably in TF 66 and further increased in Jaguar 3 brand
after 20 d treatment compared to the control plants (Figure
6C). Superoxide dismutase (SOD) activity increased at 10
d stress and then decreased at 20 d treatment compared to
that in control cultivars for both plants (Figure 6D). Com-
pared to TF 66, Jaguar 3 brand had higher APX and SOD
activities under normal or stressed regimes (P<0.05).
DISCUSSION
Photosynthesis, one of the most heat sensitive pro-
cesses, shows heat inhibition of both stomatal limitation
Table 2. Average parameters of A-Ci curves for two tall fescue (Festuca arundinacea) cultivars (Jaguar 3 brand and TF 66) grown
under normal and high temperature conditions.
Jaguar 3 brand
TF 66
Control
10 d stress 20 d stress
Control
10 d stress 20 d stress
A
max-CO2
16.60±0.75
a
14.36±0.47
b
12.62±0.18
c
14.19±1.58
a
12.38±0.45
b
11.43±0.25
c
A
sat-CO2
11.53±0.68
a
11.26±0.29
a
9.16±0.63
b
11.05±0.47
a
9.14±0.36
b
8.89±0.42
b
Rd
2.88±0.34
b
4.70±0.20
a
4.77±0.28
a
4.22±0.24
c
4.84±0.05
b
6.03±0.34
a
CE
0.043±0.001
b
0.047±0.002
a
0.037±0.003
c
0.042±0.003
a
0.039±0.008
a
0.032±0.003
b
Γ
15.03±0.86
c
17.18±1.15
b
24.29±1.43
a
20.15±3.88
b
22.28±2.16
b
28.97±1.25
a
These parameters include maximum net photosynthesis under maximum Ci (A
max-CO2
) (μmol CO
2
m
-1
s
-1
), CO
2
-saturated net pho-
tosynthetic rate (A
sat-CO2
) (μmol CO
2
m
-1
s
-1
), light respiration (Rd) (μmol m
-2
s
-1
), CO
2
compensation point (Γ) (μmol mol
-1
) and
carboxylation efficiency (CE). Each value is the mean ± S.E. based on six determinations.
Figure 5. Chlorophyll a+b, Chl a+b (A), chlorophyll a/b ratio,
Chl a/b (B), chlorophyll/carotenoid ratio, Chl/carotenoid (C),
variable chlorophyll fluorescence yield, Fv (D), maximum pho-
tochemical efficiency of photosystem .. in dark adapted leaves,
Fv/Fm (E) and efficiency of the open reaction centre in light,
ΦPSII
open
(F) in leaves of two tall fescue (Festuca arundinacea)
cultivars (Jaguar 3 brand and TF 66) grown under normal and
high temperature conditions. Measurements were conducted at
20 or 35 °C. Each value is the mean ± S.E. based on six determi-
nations.
pg_0006
66
Botanical Studies, Vol. 47, 2006
and non-stomatal limitation (Higuchia et al., 1999). In our
case, decreases of both Pn and g
s
(Figure 1A, B) under
high temperature stress indicated that reductions in CO
2
assimilation observed in the two plants were partly at-
tributable to stomatal limitation. The limitations to CO
2
assimilation imposed by stomatal closure may promote an
imbalance between photochemical activity at photosystem
II (PSII) and the electron requirement for photosynthesis,
leading to an overexcitation and subsequent photoinhibi-
tory damage of PSII reaction centers (Souza et al., 2004).
Moreover, no significant variations of Ci (Figure 1D) in
the two cultivars suggested the existence of a non-stomatal
limitation of photosynthesis in the two stressed cultivars.
Our findings were in agreement with the earlier report of
Higuchia et al. (1999). However, Camejo et al. (2005)
reported no stomatal limitation in tomato cultivars under
high temperature stress. WUE in the two stressed cultivars
decreased compared to the control plants. This decrease
was mainly attributed to decrease in Pn and increase in E
in the two treated cultivars. Under high temperature stress,
Jaguar 3 brand had a significantly higher transpiration rate
and a greater increase in transpiration rate than TF 66,
which resulted in a higher WUE in TF 66 than in Jaguar 3
brand.
The decrease in Φ (Table 1) in the two stressed cul-
tivars indicates that photoinhibition of photosynthesis
occurred (Osmond, 1994). In general, the occurrence
of photoinhibition signals an imbalance between light
energy absorption and utilization in PS... Under normal
conditions, chloroplasts recover from irreversible
photoinhibition (Colom and Vazzana, 2003). This
result was in agreement with an earlier report that
photoinhibition could be induced by high temperature
stress (Xu et al., 1995; Sinsawat et al., 2004). Moreover,
this finding could further explain the reductions of both
A
max-light
and A
sat-light
in our case. Heat stress also changed
LCP and LSP in the two cultivars, but differences between
Jaguar 3 brand and TF 66 were not significant. Higher
A
max-light
, A
sat-light
and Φ in Jaguar 3 brand than in TF 66
probably showed that these characteristics can be associ-
ated with heat tolerance in tall fescue cultivars.
Photosynthesis in the two plants was significantly influ-
enced by heat stress. This observation was also confirmed
by the analysis A-Ci curves. High temperature stress de-
creased A
max-CO2
and A
sat-CO2
, and increased Rd and Γ in the
two treated plants compared to the control plants (Table 2).
Reduction in CE in the two treated cultivars due to high
temperature stress indicated that reduction in Rubisco
activity by the stress in the two cultivars was a probable
reason for low Pn. Significant differences in A
max-CO2
, Rd
and Γ of the two plants indicated that these parameters
also probably characterised heat tolerance in tall fescue
cultivars.
Both the Chl a+b content and Chl/carotenoid ratio de-
creased, and the Chl a/b ratio increased in the two heat
stressed cultivars compared to the control plants (Figure
5A, B, C). Jaguar 3 brand retained a significantly higher
Chl a+b content and Chl/carotenoid ratio and a lower Chl
a/b ratio than TF 66 during high temperature stress. These
results suggested that these characteristics could be used
as indicators of heat tolerance and the physiological status
of tall fescue cultivars under high temperature stress con-
ditions. An increase in the Chl a/b ratio, resulting from Chl
b’s faster degradation, indicated a preferential decrease in
light-harvesting chlorophyll a/b-binding proteins (LHC)
associated with PS.. (LHC..) to transfer excitation energy
to the PS .. core complex (Xu et al., 1995). The preferen-
tial decrease in LHCII could reduce the risk of photooxi-
dative damage due to a relative decrease in the absorption
cross-section of photosystems (.pundova et al., 2003). A
decrease in LHC.I in the two stressed cultivars was attrib-
uted to more pigment composition of the photosynthesis
apparatus converting to sun-type chloroplast, which pos-
sesses less LHC... Sun-type chloroplasts are known to
possess a higher carotenoid content on a Chl basis than
medium-light or low-light chloroplasts (Camejo et al.,
2005), which could explain the lower Chl/carotenoid ratio
in the two stressed plants.
High temperature stress reduced the Fv and Fv/Fm
ratio, as well as ΦPSII
open
(Figure 5D, E, F), indicating that
a structural and functional disorder of the photosynthetic
apparatus and damage to the PS.. had occurred (Osmond,
1994; Pereira et al., 2000; Murkowski, 2001). Decrease
in Fv indicated a reduction in the number of open PSII
units. Reductions in the Fv/Fm ratio and ΦPSII
open
under
high temperature stress suggested an important portion of
the PSII reaction centre was damaged in the two stressed
cultivars. These damages were associated with structural
modifications on PSII, especially in D1 protein, which in
conditions of heat stress was phosphorylated and degraded
afterwards (Asada et al., 1998). Jaguar 3 brand had higher
Fv and Fv/Fm ratios and ΦPSII
open
during high temperature
stress than TF 66 did, indicating that the photosynthetic
Figure 6. Malondialdehyde, MDA (A), electrolyte leakage, EL
(B), ascorbate peroxidase activity (mmol mg
-1
protein min
-1
),
APX (C) and superoxide dismutase activity (Unite mg
-1
protein),
SOD (D) in leaves of control and stressed plants of Jaguar 3
brand and TF 66. Measurements were conducted at 20 or 35°C.
Each value is the mean ± S.E. based on four determinations.
pg_0007
CUI et al. — High temperature on tall fescue
67
apparatus in TF 66 was more susceptible to heat stress
than in Jaguar 3 brand.
Moreover, reduction in the Fv/Fm ratio also suggested
the occurrence of photoinhibition, also known as pho-
todamage (Colom and Vazzana, 2003). When this is the
case, accumulation of reduced electron acceptors may
increase the generation of reactive radicals such as active
oxygen species (AOS), which can induce oxidative inju-
ries (Souza et al., 2004). These oxidative injuries could
enhance Chl degradation or the inhibition of its biosyn-
thesis (Papadakis et al., 2004), damage PS.. components
(Souza et al., 2004), inactivate many chloroplast enzymes,
especially those participating in CO
2
assimilation (Dekov
et al., 2000), and could further explain the reductions
in Pn, Fv/Fm, and leaf Chl pigment content in the high
temperature stressed plants in the present study.
The occurrence of photooxidative damage, mainly
caused by active oxygen species (AOS), was further sup-
ported by significantly higher MDA and EL levels in heat
stressed plants (Figure 6A, B). MDA, a product of the
peroxidation of unsaturated fatty acids in phospholipids,
and EL have been used as indicators of free radical
damage to cell membranes and membrane thermostabil-
ity under heat stress (Lin and Kao, 1998; Liu and Huang,
2000). Our results suggest that high temperature stress
significantly increased membrane lipid peroxidation and
decreased membrane thermostability in both cultivars, es-
pecially in heat-sensitive TF 66.
Along with the occurrence of oxidative damage dur-
ing heat stress, the two plants responded by activation of
antioxidant enzymes (Figure 6C, D). Variations of APX
and SOD activities were different in Jaguar 3 brand and
TF 66 under stressed conditions. In comparison to the
control plants, activities of APX and SOD increased in the
two plants after 10 d treatment, then decreased after 20 d
treatment, except for a further increase of APX in Jaguar 3
brand. This suggests that the two stressed plants had an ef-
fective system for detoxifying active oxygen species at 10
d treatment, and this system gradually deteriorated after
20 d. Jaguar 3 brand had higher APX and SOD activities
compared to TF 66. This result, along with the remarkable
increase of MDA and EL levels, indicates that cell mem-
branes in TF 66 were more susceptible to heat stress than
in Jaguar 3 brand and that AOS scavenging ability in TF
66 was lower than in Jaguar 3 brand.
In conclusion, comparison of two tall fescue cultivars
with different heat susceptibility permitted determining
that the tolerance or sensitivity to high temperature was
manifested throughout the photosynthetic activity. High
temperature induced chlorophyll content deterioration and
functional damage in PS II and further decreased photo-
synthetic activity. Maintenance of high photosynthetic
activity is very important in enabling tall fescue cultivars
to overcome high temperature. Stomatal and non-stomatal
limitations and photoinhibition increase, as well as Rubis-
co activity decrease, also provoked CO
2
assimilation rate
reduction in the two stressed cultivars. The function of PS
II and the pigment content of the light harvesting com-
plex were important aspects of the tolerance of tall fescue
plants to high temperature. The preservation of higher
antioxidant ability to curtail lipid peroxidation and cell
membrane damage was also associated with the heat toler-
ance of tall fescue cultivars.
Acknowledgements. We are grateful to Judy Hunter
(Writing Lab, Grinnell College, Grinnell, Iowa, USA) for
critical reading of the manuscript. We are also grateful
to Professor Liang Zongsuo (Institute of Soil and Water
Conservation, Chinese Academy of Sciences and Ministry
of Water Resources, Yangling, P.R. China) for suggestions
on the work. Illuminating comments from the chief editor
and two anonymous reviewers are also appreciated. The
research was supported by the Nanjing University Science
Fund.
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高溫脅迫對兩個耐熱性不同的高羊茅品種光合作用，
光系統 II 功能和抗氧化活性的影響
崔浪軍　李建龍　范亞民 徐　勝　張　真
中國江蘇南京大學生命科學學院
　　
高溫是高羊茅草坪草 (Festuca arundinacea) 在亞熱帶地區夏季生長的主要限制因數。本文以高羊茅
草坪草耐熱型 "美洲虎 3 號" 和熱敏感型 "TF 66" 的葉片為材料，比較研究了高溫脅迫對高羊茅兩個品
種的光合作用、光系統 II 功能和抗氧化活性的影響。結果表明，高溫脅迫通過誘發了葉片氣孔限制和
非氣孔限制，促使光抑制增加和 Rubisco 活性下降而降低了高羊茅的淨光合速率 (Pn)。高溫 脅迫改變了
高羊茅葉片光系統 II 的功能，表現為高溫脅迫下兩品種的葉片可變熒光 (Fv)、光系統 II 最大光化學量
子產量 (Fv/Fm) 以及光下開放反應中心效率 (ΦPSII
open
) 都顯著降低。高溫脅迫極大地降低了兩品種葉綠
素 a+b 含量和葉綠素/類胡蘿½素比率，增加了葉綠素 a/b 比率。 此外，兩供試品種受脅迫後葉片½脂
過氧化程度顯著增加，細胞½熱穩定性顯著降低，抗壞血酸過氧化物. (APX) 活性和超氧化物岐化.
(SOD) 活性也發生了明顯的改變。相同程度的高溫脅迫對 "TF 66" 的光合作用，光系統 II 功能和抗氧化
活性的影響比對 "美洲虎 3 號" 的影響更大。本研究結果為日後進行耐熱型高羊茅品種的選育工作提供
了一些新的指導。
關鍵詞：抗氧化.；細胞½完整性；高羊茅；高溫脅迫；光合特性；光系統 II 光化學效率。
pg_0010