Current physiological parameters used for selection of tolerant and sensitive genotypes to environmental stresses are generally costly and time-consuming. Therefore, it is necessary to achieve physiological criteria that would be accurate and inexpensive, despite eliminating the aforesaid disadvantages. Accordingly, 11 various lines of lentils were cultivated and grown using the hydroponic system. At 4-5 leaf stage, lentil seedlings were exposed to salinity stress for 10 days by adding 50 and 100 mM sodium chloride to a nutrient solution. Afterward, the chlorophyll a fluorescence in lentil seedlings was evaluated in normal conditions and the mentioned salinity stress. In this experiment, several parameters were measured, including fluorescence at 50, 100, 300 µs, 2 and 30 ms, Fm, variable fluorescence (Fv = Fm-Fo), relative variable fluorescence at J phase (VJ = (FJ – Fo)/(Fm – Fo)), variable fluorescence per maximum fluorescence (FV/Fm), the area under the curve (Area) of OJIP and performance index (PI). The results showed that three lines had higher photosynthetic efficiency than other lines.
Keywords: chlorophyll a fluorescence, Lens culinaris, OJIP curve, salinity stress
Salinity is one of the most important abiotic stresses that could affect the physiological and biochemical processes of the plant by disturbing the ionic balance and plant-water relations. Reduction in photosynthetic efficiency is one of its main effects (Sayed, 1999; Munns, 1993; Mishra et al., 1991; Flowers et al., 1977). Salinity reduces the photosynthesis by affecting the electron transport chain in light-dependent reactions (Parihar et al., 2015; Borsani et al., 2001). Within the occurrence of salinity stress and limitation of carbon dioxide (because of osmotic potential of plant cells and stomatal closure), emission of light photons on the leaves continues and chlorophyll gets stimulated by absorbing the energy of photons. In order to prevent deterrence of light and oxidative stress, the stimulated chlorophyll should become stable. One of the methods to deal with salinity is the use of biological methods in which plants get modified for salt tolerance trait by the help of physiological information and selection criteria (Munns and Tester, 2008; Munns, 2002). Salinity extremely decreases the activity of photosystem II, and dysfunction of quinones electron transport chain in photosystem II reduces the quantum yield (Gonçalves et al., 2007; Lu and Vonshak, 2002).
Generally, the chlorophyll a fluorescence is a reliable physiological indicator to determine changes in the induced photosynthetic apparatus (Mehta et al., 2010). Assessment operations of this indicator are being performed in the shortest time without the destruction of plant tissue. In addition, a large number of genotypes can be evaluated in a short time using this technique (measuring the chlorophyll a fluorescence) (Belkhodja et al., 1994). The chlorophyll a fluorescence is a multi-stage sign that is called O-J-I-P curve (Strasser et al., 1995). The O-J-I-P curve shows the consecutive reduction or oxidation of electron transfer in photosystem II (Govindjee, 1995). The OJIP test is a developed method to introduce the different parts of O-J-I-P curve (Mehta et al., 2010) that is recorded in a specific time, so that O is the fluorescence intensity at 50 ms, J is the fluorescence intensity at 2 ms, I is the fluorescence intensity at 30 ms and P is the Fm at a specified time (Zhang et al., 2015; Oukarroum et al., 2007). Moreover, in this curve, FL represents the fluorescence at 100 µs and FK indicates the fluorescence at 300 µs (Wang et al., 2010; Gonçalves et al., 2007; Strasser et al., 2000). Strasser et al. (2004) reported that fluorescence measured at 100 to 200 µs was the L-band and fluorescence measured at 200 to 400 µs was the K-band (Oukarroum et al., 2007). Oukarroum et al. (2007) considered the fluorescence measured at 150 µs as the FL and fluorescence measured at 300 µs as the FK. The variable fluorescence (FV = Fm-Fo), variable fluorescence per maximum fluorescence ratio (FV/Fm) and relative variable fluorescence at J phase (VJ = (FJ – Fo) / (Fm – Fo)) are some of the other key parameters calculated in the chlorophyll fluorescence. In the OJIP curve, the area under the curve (Area) shows the size of the quinones pool of electron acceptor (QA, QB, and PQ) (Kato et al., 2016; Joliot and Joliot, 2002).
In the Fo, all reaction centers are open, so the maximum excited energy level can be utilized. In this case, most of the energy of molecules at its excited state is used in the photochemical reaction, and the fluorescence is minimized. Rapid increasing of Fo is because of the damage to electron transport chain of photosystem II, as a result of the capacity reduction of QA and lack of complete oxidation, caused by electrons flow along the photosystem II (Zlatev and Yordanov, 2005; Krause and Weis, 1991).
FJ represents the oxidized form of assembly carriers of photosystem II, including P680, as well as the reduced form of the carrier QA (QA-). FI refers to the reduction state of electron acceptor of the photosystem II, involving QB and QA carriers (Lazár, 1999; Govindjee, 1995). All the electron carriers are reduced due to the exposure of photons radiation and all reaction centers are closed; at this time, fluorescence has the maximum level (Fm) (Mehta et al., 2010; Strasser et al., 2000). The maximal fluorescence during the stress condition is decreased because of the reduction in activity of degrading complex and photosystem II as well (Nath et al., 2013; Aro et al., 1993). The Fv is calculated by subtracting the Fm and the fluorescence at 50 µs (Fm – Fo). FV demonstrates the electron flow from donor section of photosystem II to the QA (Xia et al., 2004).
FV/Fm ratio is the other important parameter calculated in chlorophyll fluorescence. FV/Fm shows the maximum quantum efficiency of photosystem II for converting absorbed light into chemical energy. The decrease in FV/Fm ratio is the result of damage to the reaction centers of photosystem II, which can reduce the maximum quantum efficiency of photosystem II (Baker and Rosenqvist, 2004; Kocheva, 2004). The amount of fluorescence at the J, P and I is decreased in salinity condition, which may be for two reasons: the electron transfer in photosystem II which is due to the accumulation of oxidized state of P680 (Hasegawa et al., 2000), and the reduced size of QA (Mehta et al., 2010).
The most important parameter in OJIP test is PI. PI parameter converts the three factors involved in photosynthesis operating procedures into a multivariate factor, including the number of reaction centers in the stroma of chlorophyll, the level of the trapping excited energy and the conversion rate of excited energy to electron transfer (Živ?ák et al., 2008; Strasser et al., 2004; Tsimilli-Michael, et al., 2000). In addition to indicating the fluorescence yield at the end of Fo and Fm, PI can also demonstrate the fluorescence at their intermediate points such as the J phase. Furthermore, the slope of fluorescence can be also detected by the means of this index (Strauss et al., 2006). PI provides the possibility of comprehensive analysis of photosynthetic performance. The relationship between the energy capture in photosystem II and the efficiency of photon absorption, the density of reaction centers and the possibility of electron transfer to after the QA- by the excited energy can be investigated using this parameter (Dabrowski et al., 2016; Živ?ák et al., 2008; Gonçalves et al., 2007).
MATERIALS AND METHODS
Lens culinaris, lentil, is an annual herbaceous plant in the Fabaceae (legume or bean family) that is considered to be one of the world’s oldest crops. The experiment was performed on 11 lines of lentil (Table 1) that were prepared from the Moghan Agro-industrial ; Livestock Company at three levels of control level (0 mM) with salinity level of 50 mM and 100 mM of sodium chloride as the factorial experiment based on completely randomized design with three replications. Before sowing, the seeds were disinfected with sodium hypochlorite 5% to prevent contamination of the plant by fungi during growth. Then, the seeds were soaked and germinated in distilled water in Petri dishes for 72 hours (at 25°C and low light). When the shoot length reached approximately 1 cm, the seedlings were cultured in a depth of 2 cm in hydroponic culture. Hydroponic culture medium contained Perlite and peat moss at a ratio of 1:3. The lentil seedlings were grown to 4-5 leaf phase in the complete nutrient solution. After this phase, salinity stress at 50 and 100 mM/L were applied using sodium chloride. The plants were kept for 10 days under the same conditions.
The chlorophyll a fluorescence was measured using Handy PEA device (Hansatech, UK). The selected leaves were put in a place with no light for 15 minutes by mean of the special clips. This time was determined by different tests times. In this dark period, all the reaction centers are wide-open. Then, a pulse of light at a wavelength of 650 nm with an intensity of 3000 µM photons per m2/s was applied to the leaves for 4 seconds. Then, OJIP parameters of variable fluorescence values at the time of 50 µs, 2 and 30 ms were recorded using special software of the devices (Handy PEA Software V1.30, 2001), respectively, indicating the amount of fluorescence in mentioned times (Strasser et al., 2000).
Normal distribution of data and errors test was performed via SPSS software before statistical analysis and analysis of variance. Then, the data in the factorial experiment format based on completely randomized design were analyzed by SPSS software, ANOVA and Duncan’s multiple range tests to compare the means.
RESULTS AND DISCUSSION
The analysis of variance results of salinity interaction on OJIP curve indices as the factorial experiment based on completely randomized design is given in Table 2 regarding the studied lines. The results of analysis of variance showed that the salinity effect, cultivar effect and interaction of salinity on the cultivar based on fluorescence parameters in 50 µs or in O phase, fluorescence at 100 µs or in L phase, fluorescence at 300 µs or in K phase, fluorescence at 2 ms or in J phase, fluorescence in 30 ms or in I phase, variable fluorescence (Fv), Fm, variable fluorescence per maximum fluorescence (Fv/Fm), relative variable fluorescence at J phase (VJ), area under the curve and PI were significant at the level of 1%.
The mean comparison of salinity interactions with cultivar on the OJIP curve parameters
According to the results of analysis of variance, due to the significance of salinity interactions with the cultivar, the mean comparison was performed using this variable by Duncan’s multiple range tests at the level of 5%.
The results of the mean comparison of fluorescence at 50 µs indicated that the studied lines were divided into two groups of A and B under experimental treatments. The lines of group A under salt stress had no significant changes in the level of their fluorescence compared to the control. Lines 2, 9 and 11 were placed in this group. Lines 1, 3, 4, 5, 6, 7, 8 and 10 were placed in the group B that their level of fluorescence had significant reduction compared to the control. In the salinity level of 50 mM, the maximum and minimum decreases were respectively related to lines 9 and 10 compared to the control. At the salinity level of 100 mM, the highest and lowest decreases were related to the line 10 (Fig. 1). All reaction centers are open at the time of fluorescence measurement at 50 µs, so most of the energy of the excited molecules is used at the photochemical reaction and less energy is wasted in the form of fluorescence. At salinity stress, fluorescence level at 50 µs is reduced in wheat (Mehta et al., 2010). Xia et al. (2004) have reported no significant difference at Fo in Ulva lactuca.
The results of mean comparison demonstrated the reduction in FL level at all lines caused by experimental treatments. The Fm reduction at the salinity level of 50 mM was related to the line 10 with a 43% reduction compared to the control and the less reduction was associated with the line 11 with 2% reduction. At the salinity level of 100 mM, the maximum and minimum reductions were respectively found in line 10 with 44% and line 11 with 4% (Fig. 2). Strasser et al. (2004) considered the measured fluorescence at the intervals of 100 to 200 µs as L band. The decrease in fluorescence at 100 µs represents the functional limitation of the electron donor in the photosystem II. Researchers investigated the barley cultivars and observed that the fluorescence at this phase is decreased under the stress condition. The fluorescence is under the influence of electron transfer by photosystem II components at this phase (Oukarroum et al., 2007).
The results of the mean comparison indicated that the FK was reduced in the lentil lines affected by salinity stress. Line 10 (33%) out of the lines at the salinity level of 50 mM had the maximum reduction compared to the control and the lowest reduction was related to the line 11 (0.5%). The minimum reduction was seen in line 20 with 4% reduction (Fig. 3). Strasser et al. (2004) considered the measured fluorescence at the intervals of 200 to 400 µs as K band. The incidence of fluorescence at K-phase is caused by an imbalance between the reaction center of the electron acceptor and electron donor section in the photosystem II.
The results of the mean comparison of FJ showed that lines A and B were divided into two groups. Group A includes the lines in which the fluorescence intensity at J phase had no significant reduction compared to the control. Lines 2, 9 and 11 were in this group. Group B includes the lines in which the fluorescence at J phase had reduction compared to the control. Lines 1, 3, 4, 5, 6, 7, 8 and 10 were placed in this group (Fig. 4). In a study about the effect of cold on soybean, Strauss et al. (2006) reported a decrease in fluorescence at this stage due to cold. At this phase, under saline conditions, fluorescence reduction in wheat also has been reported (Mehta et al., 2010).
According to the results of the mean comparison in all lines, FI significantly was decreased due to salinity stress. At the salinity level of 50 mM, Line 3 with 0.2% reduction had the lowest decrease in fluorescence and Line 4 with 9% reduction had the highest fluorescence reduction compared to control. At the salinity level of 100 mM, the highest decrease in fluorescence was related to the line 5 with 22% compared to the control level and the lowest reduction was related to the line 6 at the rate of 2% (Fig. 5). Fluorescence at the 30 ms represents the reduction state of the electron acceptor in the photosystem II that may be mentioned to QA and QB carriers (Govindjee, 1995). The decrease in fluorescence has also been reported at this phase in wheat under salinity stress conditions (Mehta et al., 2010).
The results showed that the lines under the influence of experimental treatments were divided into two groups A and B in terms of reduction in the level of Fm. Group A included lines 2, 9 and 11 in which reduction in the level of the Fm was not significant. The lines 1, 3, 4, 5, 6, 7, 8 and 10 were allocated to the group B in which the effect of the decline of Fm on the salinity stress was significant. In the salinity level of 50 mM NaCl, the greatest reduction in Fm was related to the line 10. Line 11 had the lowest level of reduction compared to the control group. In addition, line 10 and line 11 had respectively the greatest and lowest reduction of Fm in the salinity level of 100 mM NaCl (Fig. 6). Because of the photons emitted, all electron carriers change to reduce state and all reaction centers are closed. Chlorophyll a indicates the Fm at this time (Mehta et al., 2010). The researchers reported a reduction in the Fm of algae Ulva lactuca in salinity stress. These researchers believe that reduced Fm is related to the accumulation of oxidized state of P680 and a decline in the capacity of reduced QA quinones or plastoquinones (PQ) )Xia et al., 2004).
The lines were divided into two groups A and B based on the Fv changes in salinity stress. Group A includes the lines in which Fv intensity did not significantly change under the effect of the experimental treatments. Lines 2, 9 and 11 were placed in this group. Group B contains the lines in which the mentioned fluorescence had no significant change compared to the control. Lines 1, 3, 4, 5, 6, 7, 8 and 10 were subjected in this group (Fig. 7). The Fv was obtained from the difference between the Fm and Fo. Majority of Fv represents a good performance of chlorophyll fluorescence mechanism under stress conditions and reduction photochemical reactions (Sarkar and Ray, 2016; Stirbet et al., 2014; Baker and Rosenqvist, 2004).
The results showed that the Fv per Fm ratio under the salinity stress is reduced in all of the studied lines. However, this reduction was not significant in lines 2, 9 and 11, but it was significant in the rest of the studied lines. In salinity level of 50 mM NaCl, line 2 with less than 0.1 % reduction had the lowest decline and Line 5 with 4 % reduction had the greatest decline compared to the controls. In salinity level of 100 mM NaCl, compared to the control level, the highest decrease of FV/Fm was related to the line 5 with a rate of 9% and the lowest reduction was related to the Line 2 with a rate of less than 0.5%. The mean FV/Fm ratio between the salinity level of 50 and 100 mM NaCl was not statistically significant for lines 2, 9 and 11 (Fig. 8). The FV/Fm ratio indicates the quantum efficiency of photosystem II to convert the absorbed light into chemical energy (Mancarella et al., 2016; Stirbet and Govindjee, 2011). In wheat under salinity stress, this index was significantly decreased. Reduction of this ratio indicates the damage to the reaction centers of photosystem II (Azizpour et al., 2010). Belkhodja et al. (1994) reported that salt-tolerant varieties of barley have higher values of Fv/Fm ratio than the susceptible cultivars. In other words, the efficiency of photosystem II was higher in resistant varieties.
The results of the mean comparison of VJ indicated that the lentil lines were divided into three groups of A, B and C. Group A included the line 9 which its VJ was increased compared to the controls. Group B consists of lines which had a significant reduction in VJ compared to the controls; and lines 1, 3, 4, 5, 6, 7, 8 and 10 were allocated to this group. Group C included the lines 2 and 11 with no significant change in VJ compared to the controls (Fig. 9). VJ indicates the ratio of the first quinones of the reduced electron acceptor (QA-) to the total available quinones in photosystem II (Strasser et al., 2000; Strasser et al., 1995). Force et al. (2003) stated that VJ demonstrates the ratio of the number of closed reaction centers on the J phase to the total number of the closed reaction centers. The lack of the change in VJ in salinity stress shows that electron donor section and QA are not affected by salinity, and the increase in VJ salinity stress represents the accumulation of QA-, which has been reported in wheat (Mehta et al., 2010; Wang et al., 2010).
The area under the curve of OJIP was reduced in all studied lines under the effects of salinity. The mean area under the curve between salinity levels of 50 and 100 mM NaCl for lines 2, 9 and 11 were not statistically significant. The highest decrease in the salinity level of 50 mM was related to the line 4 that had 21% reduction compared to the controls. Line 11 with less than 1% had the lowest reduction compared to the level of the control group. In the salinity level of 100 mM, Line 4 with 52% reduction had the greatest decrease compared to the control group, and line 11 with more than 4% had the lowest decline (Fig. 10). The area under the curve of OJIP shows quinones pool volume of the electron acceptor. Reduced quinones pool volume is considered as a constraint because of quinines, especially PQ act as a mediator in electron transfer from photosystem II to photosystem I (Stirbet et al., 2014; Stirbet and Govindjee, 2012; Gonçalves et al., 2007; Strasser et al., 2004). Drop in this index could cause sensitivity to environmental stresses such as salt. Reduction of this index under salinity stress in wheat has been reported (Mehta et al., 2010).
Lentil lines were divided into three groups of A, B, and C in terms of the PI. Group A included Line 11 in which the PI was increased compared to the controls. Group B involved lines with no significant change in this parameter; and lines 2 and 9 were allocated to this group. Group C included the line that had the significant reduction in PI compared to the controls; and lines 1, 3, 4, 5, 6, 7, 8 and 10 were in this group (Fig. 11). PI is an appropriate indicator for evaluating the performance of plant to absorb light energy, energy capture and conversion of energy excited to electron transfer by photosynthesis under environmental stresses such as salinity, drought, and heat. PI provides a comprehensive analysis of photosynthetic performance. Deactivation of a large number of reaction centers on the leaf surface, high intensity of light energy, reduction in the parameter of the maximum quantum performance of photosystem II and the flow of the electrons after QA- are some of the parameters that lead to reduction in the PI (Dabrowski et al., 2016; Mehta et al., 2010; Živ?ák et al., 2008).
The purpose of the current study was to determine the resistant lines to salinity stress by using chlorophyll fluorescence. The results of biophysical parameters showed that lines 2, 9 and 11 had higher photosynthetic efficiency than other lines because of less declining of parameters such as area, Fv/Fm ratio and PI under salt stress conditions compared to control. It is suggested that the association of these parameters and the level of expression of relevant genes is investigated in future studies.