2.3.2 Methyl viologen (MV)
MV (0.5 µl, 2 h) accepts electrons from PSI and limits the electron flow
to CEF. Second detached leaves of wheat seedlings were infiltrated with
0.5 µl MV for 2 h before stress.
Measurement of oxygen evolution rate
According to the method described in our previous study (Luo, Li, Wang,
Yang & Wang, 2010), the oxygen evolution rate was measured with a
Clarke type O2 electrode unit (Hansatech, King’s Lynn,
UK) in the thylakoid membranes. The results were the average of 5
independent replicates.
Thylakoid membrane proteins extraction and quantification
Thylakoid membranes were prepared in accordance with the method of
Rintamaki et al. (1996). Winter wheat leaves were ground and homogenized
with cool isolation buffer. The homogenates were centrifuged at 1500g for 4 min at 4°C. The pellets were washed with 10 mM
HEPES-NaOH, pH 7.5, 5 mM sucrose, 5 mM MgCl2, and 10 mM
NaF and pelleted at 3000 g for 3 min. Thylakoid pellets were
resuspended in a small amount of storage buffer, and then were stored at
-80ºC before use.
SDS-PAGE and western blot analysis
According to experimental method of Du et al. (1995), thylakoid membrane
proteins were separated using 15% polyacrylamide gel. In total, 5 μg of
chlorophyll was loaded per lane. The resolved proteins were transferred
to NC membrane from gel and detected using a D1 protein antibody
(Agrisera AB, 1:5000). Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 680)
(Abcam, 1:10000) was used as the secondary antibody. Infrared laser
imaging system (Odyssey CLx, USA) was used to detect the membrane and
the relative content of D1 protein was obtained by Image J software.
Measurement of PQ Pools
The P700 signal was determined during single turnover flashes (ST, 50ms,
PQ pools being oxidized) followed by multiple turnover flashes (MT 50
ms, PQ pools are fully reduced) in the presence of far-red background
light (Savitch et al., 2001). The complementary area between the
oxidation curve of P700 after single turnover and multiple turnover
excitation and the stationary level of P700+ under
far-red represents the single turnover - and multiple turnover-areas,
respectively. These were used to calculate the functional pools sizes of
intersystem electrons relative to P700 as follows:
e-/P700 = multiple turnover-areas/single turnover
-areas (Savitch et al.,2001).
P515/P535 measurements
With the experimental method by Schreiber and Klughammer (Schreiber &
Klughammer, 2008) as reference, the dual-beam 550 nm to 515 nm
difference signal was monitored simultaneously by using the P515/535
module of the Dual-PAM-100 (HeinzWalz, Effeltrich, Germany). After 10
min of pre-illumination at 531 µmol quanta m−2s−1 and 4 min of dark adaptation, P515 changes induced
by saturating single turnover flashes were recorded to evaluate ATPase
activity. Slow dark-light-dark induction transients of the 550 nm to 515
nm signals reflect changes in the membrane potential (electrochromic
pigment absorbance shift). After 30 s, actinic light (AL; 531 µmol
quanta m−2 s−1) was turned on and
off after 330 s.
Statistics
All graphs were made using Origin 8.0 software (Origin Lab, Northampton,
MA, USA). Statistical analyses were performed by ANOVA using SPSS
version 21.0 (SPSS, Chicago, USA), and comparisons between the mean
values were accomplished by the least significant difference test at the
0.05 probability level. Quantitative assessment was conducted on
randomly selected samples from five independent biological replicates.
3 RESULTS
Effects of trehalose pretreatment on changes in the initial
reduction rate of P700+ under heat and drought
stress
A higher initial P700+ reduction rate was observed in
trehalose pretreated seedlings under heat and drought stress compared
with control plants (Figure 1). After a 24 h recovery from drought
stress, the enhancement effect of the initial reduction of
P700+ by trehalose pretreatment was retained. However,
no significant difference was detected in the effect of the initial
P700+ reduction rate by exogenously supplied trehalose
between the control group and the stressed group after a 24 h recovery
from heat and drought plus heat stress.
Figure 1 shows no differences in the initial P700+reduction rate between trehalose-pretreated and control seedlings during
the 24 h recovery (R2 and R3).
Effects of trehalose pretreatment on changes in D1 protein
content under heat and drought stress
Western blot was used to determine whether the D1 protein was affected
by trehalose under heat and drought stress. D1 protein content increased
compared to the control in response to exogenously supplied trehalose
during drought and drought plus heat stress (Figure 2B1). When leaves
were incubated with SM, a D1 protein synthesis inhibitor, a lower D1
protein content was obtained in seedlings without trehalose than in
trehalose pretreated plants under drought stress (Figure 2B2).
Effect of trehalose pretreatment on changes in Fv/Fm under
heat and drought stress
Photoinhibition of PS II was measured by comparing the photochemical
efficiency values to further study the role of trehalose in the D1
protein and PS II. The photochemical efficiency values in the control
and trehalose-pretreated seedlings
decreased after the plants were
heat and drought stressed (Figure 3). A higher photochemical efficiency
value was observed in the trehalose pretreated seedlings than the
control plants. Seedlings without trehalose suffered more severe
photoinhibition than
trehalose-pretreated plants under heat and drought stress when leaves
were incubated with SM.
Effect of trehalose pretreatment on changes in the oxygen
evolution rate under heat and drought stress
The oxygen evolution rate
decreased in the control wheat
seedlings under heat and drought stress (Figure 4). A higher oxygen
evolution rate was detected in the trehalose pretreated seedlings than
that in control plants.
Effect of trehalose pretreatment on changes in the electron
transport rate of PS II (EFR(II)) under heat and drought stress
EFR(II) was significantly lower under drought and heat stress
compared to that of the control plants (Figure 5). The trehalose
pretreatment improved EFR(II) significantly under drought and
heat stress.
Effect of trehalose pretreatment on changes in thePQ pool under heat and drought stress
The PQ pool decreased in control
wheat seedlings under heat and drought stress (Figure 6). The trehalose
pretreated seedlings had a higher PQ pool than the control plants.
Effect of trehalose pretreatment on changes in ATPase activity
under heat and drought stress
Figure 7 shows the rapid decay of the P515 signal after illumination.
Faster decay of the P515 signal represents higher ATPase activity.
Higher ATPase activity was observed in trehalose pretreated seedlings
than control plants under heat and drought stress. Lower ATPase activity
was observed in seedlings without trehalose than in trehalose pretreated
plants when leaves were incubated with MV under heat and heat plus
drought stress.
Effects of trehalose pretreatment on changes in ΔpH across the
thylakoid membrane under heat and drought stress
ΔpH component of the proton motive force (ΔpH/pmf) increased
significantly in control wheat seedlings under heat and drought stress
(Figure 8). A higher ΔpH/pmf was observed in trehalose pretreated
seedlings than in control plants. A higher ΔpH/pmf was observed in
seedlings with trehalose than control plants when leaves were incubated
with MV under heat and heat plus drought stress.
4. DISCUSSION
PS II performance of wheat leaves under heat and drought
stress
Our results showed that heat and drought stress caused reversible
photoinhibition of PS II (Figure 3). Blocked linear electron transport
(Figure 5) resulted in a potential excess of light excitation pressure
in the PS II reaction center after heat and drought stress. Excess
energy in PS II can lead to the generation of reactive oxygen species,
which are deleterious to the function and structure of PS II (Liu, Qi &
Li, 2012). In this study, the oxygen evolution rate decreased in the
control wheat seedlings under heat and drought stress (Figure 4),
indicating that OEC may have been damaged. Destruction of the OEC and D1
protein damage have detrimental effects on the PS II reaction center
(Wang, Wang, Hu, Chang, Bi & Hu,2015). Our results also show that D1
protein content was significantly lower than that of the control under
heat and drought stress (Figure 2B1), indicating destruction of the PS
II reaction center. Furthermore, stress blocked electron transfer
(Figure 5) and reduced photochemical efficiency (Figure 3).
Subsequently, a low oxygen evolution rate was obtained (Figure 4). These
results are evidence of PS II damage.
Fortunately, plants have developed various photo-protective mechanisms,
such as CEF, to alleviate damage to PS II. Plants adapt to a variety of
environmental stressors by stimulating CEF (Hare, Cress & Van
Staden,1998) As CEF generates ΔpH across the thylakoid membrane
(Munekage et al.,2002 ) by transferring electrons from PSI to PQ, it is
important to protect PS II by dissipating excess light energy(Takahashi,
Milward, Fan, Chow & Badger, 2009). Our results show that CEF was
stimulated (Figure 1) and ΔpH increased significantly (Figure 8) under
heat and drought stress. In addition, as the functional PQ pool was
significantly inhibited by the heat and drought treatment compared to
the control (Figure 6), the decrease in PQ was the major factor blocking
electron transport (Figure 5). A reduction in the PQ pool decreases PS
II excitation, increases CEF, alleviates the ATP deficit, and increases
ΔpH thereby downregulating the PS II antenna via the qE mechanism (Yi,
Mcchargue, Laborde, Frankel & Bricker,2005). Our results also show that
ATPase activity was significantly higher than that of the control under
heat and drought stress (Figure 7).
The PS II protective effect of trehalose under heat and
drought stress
Some recent studies have demonstrated that exogenous trehalose is
effective for protecting the PS II complex under stress conditions. For
example, trehalose increases the electron transfer rate of PS II in
Mn-depleted PS II membrane fragments of spinach (Yanykin, Khorobrykh,
Mamedov & Klimov, 2015). In addition, trehalose significantly
stimulates and stabilizes the oxygen evolution rate in the PS II complex
(Mamedov, Petrova, Yanykin, Zaspa & Semenov, 2015). Our results show
that the trehalose pretreatment increased the PQ pool under heat and
drought stress (Figure 6). The increase in the PQ pool may be
responsible for the higher linear electron transport observed in the
trehalose-pretreated groups compared with the control (Figure 5), in
agreement with an earlier study (Zhang, Liu, Ni, Meng, Lu & Li, 2014).
In the present study, the trehalose pretreatment significantly promoted
CEF under heat and drought stress (Figure 1). The increase in CEF was
essential for the higher ΔpH across the thylakoid membrane and ATPase
activity in the trehalose-pretreated seedlings compared with the control
plants (Figure 7 and 8). These results show that the trehalose
pretreatment improved ΔpH and ATPase activity by promoting CEF under
heat and drought stress.
Our results show that the trehalose pretreatment increased D1 protein
content and the oxygen evolution rate under heat and drought stress
(Figure 2 and 4). ΔpH depends on CEF to play a key role in protecting
the OEC in Dalbergia under high light intensity (Huang, Yang, Hu,
Zhang & Cao, 2016). CEF provides energy to repair the D1 protein in the
PS II core complex by establishing ΔpH and synthesizing ATP. Therefore,
the increase in D1 protein content and the oxygen evolution rate may
have significant associations with the higher CEF observed in the
trehalose-pretreated groups than the control plants. Furthermore,
inhibition of PS II was relieved by the trehalose pretreatment (Figure
3).
In addition, the absolute rate of in vivo D1 protein degradation
can be established provided it is not affected by de novo D1
synthesis (Schnettger, Critchley, Santore, Graf & Krause,1994) Our
results show that the trehalose pretreatment increased D1 protein
content by promoting synthesis of the D1 protein under heat and drought
plus heat stress, whereas it reduced degradation of the D1 protein under
drought stress (Figure 2). Under heat and drought plus heat stress,
exogenous trehalose enhanced ΔpH and ATPase activity, which did not
entirely depend on CEF (Figure 7 and 8), and other potential mechanisms
may exist. The common physiological function of both the water-water
cycle and CEF is to supply ATP and ΔpH (Miyake, 2010). Therefore, the
other potential mechanism may be
the water-water cycle. However,
the trehalose pretreatment increased ΔpH and ATPase activity only by
stimulating CEF under drought stress (Figure 7 and 8).