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).