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Interaction of Salinity and Phytohormones on Wheat Photosynthetic Traits and Membrane Stability

Interaction of Salinity and Phytohormones on Wheat Photosynthetic Traits and Membrane Stability DOI: 10.2478/agri-2013-0004 ARMAN PAZUKI 1, MOHAMMAD SEDGHI 2, FATEMEH AFLAKI 2* University of Guilan, Rasht University of Mohaghegh Ardabili, Ardabil PAZUKI, A. ­ SEDGHI, M. ­ AFLAKI, F.: Interaction of salinity and phytohormones on wheat photosynthetic traits and membrane stability. Agriculture (Ponohospodárstvo), vol. 59, 2013, no. 1, pp. 33­41. To evaluate phytohormones effects on stomatal conductance, chlorophyll fluorescence, membrane stability, relative water content and chlorophyll content under salinity, a factorial experiment with 4 replicates was conducted. Treatments were salinity (0, 3.5 and 7 dS/m), phytohormones (control, gibberellic acid and abscisic acid) and wheat cultivars (Gascogen, Zagros, and Kuhdasht). Results showed that a high level of salinity increased chlorophyll fluorescence and relative water content, while membrane stability, chlorophyll content, and stomatal conductance were decreased. Abscisic acid treatment had more effective role in membrane stability. Although membrane stability was much more under gibberellic acid treatment, restoration of membrane stability was considerable under abscisic acid treatment for Gascogen and Kuhdasht cultivars. Spraying of gibberellic acid induced the highest chlorophyll content in the three salinity levels and all of the cultivars. The maximum amount of stomatal conductance was achieved under gibberellic acid treatment. Abscisic acid caused less chlorophyll fluorescence in comparison to gibberellic acid. About relative water content, abscisic acid was effective in high salinity levels so that it caused stomatal closure, which reduced water loss and maintained turgor in plants. Key words: Triticum aestivum, NaCl, gibberellic acid, abscisic acid, chlorophyll fluorescence, stomatal conductance Salinity is one of the major abiotic stresses affecting plant growth, development and productivity (Rahnama & Ebrahimzadeh 2004). Salinity is a complex environmental constraint that presents two main components: i) osmotic component due to decrease in the external osmotic potential of the soil solution and ii) ionic component linked to the accumulation of ions which become toxic at high concentrations (mainly Na + and Cl - ) (El-Bassiouny & Bekheta 2004). It is estimated that over 800 million hectares of land in the world are affected by both salinity and sodicity (Munns 2005). Plants exposed to salt stress must undergo changes in their metabolism in order to survive in the deleterious condition of the stress (Rahnama & Ebrahimzadeh 2004). These changes include stomatal conductance (SC), photosynthetic efficiency, water and nutrients availability (Munns & Termaat 1986). Chlorophyll content (CC), relative water content (RWC) and SC are affected by increasing salinity (Adnan Shahid et al. 2008). RWC represents a useful indicator of the state of plant water balance, because it expresses the absolute amount of water, which plant requires reaching full saturation (González & González-Vilar 2001). Leaf RWC is a useful trait for selection of tolerant plants in saline condition (Schonfeld et al. 1988) and is significantly reduced under salinity stress (El-Tayeb 2005). Furthermore, salinity stress also reduces RWC in sensitive wheat cultivars seedlings (Aldesuquy & Gaber 1993). Fatemeh Aflaki* (Corresponding Author), Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Mohaghegh Ardabili, Ardabil, Iran. E-mail: fa_aflaki@yahoo.com Chlorophyll (Chl) is one of the most important pigments, and is responsible for green colour in plants. Changes in photosynthetic parameters could potentially be used as a screening method for salinity tolerance in plants, because more tolerant cultivars are expected to exhibit less disturbances in photosynthetic processes (Belkhodja et al. 1999). Generally, Chl content is reduced under salinity conditions (Iqbal et al. 2006). One of the leading parameters for the estimation of salinity stress effects is chlorophyll fluorescence (CF). CF is a subtle reflection of primary reactions of photosynthesis (Sayed 2003). The ratio of Fv/Fm is proportional in order to show maximum amount of photosystem II (PSII), and this ratio is sensitive to environmental changes (Sayed 2003). Krishnaraj et al. (1993) used measurement of CF for screening of tolerant wheat genotypes (Krishnaraj et al. 1993). Various stress conditions may reduce the rate of photosynthesis, and disturb the light-driven photosynthetic electron transport via CF fluorescence (Allahverdiev et al. 1998). The inverse relationship between in vivo CF and photosynthetic activity can be used to study the potential photosynthetic activity of leaves and detect stress effects on green plants. There are several parameters of the in vivo CF which can be applied for detecting stress and damages to the photosynthetic apparatus. Variable fluorescence (Fv) and initial fluorescence (Fo) ratio are two of these parameters, and they show the efficiency of photosynthesis (Allahverdiev et al. 1998). The decline in productivity in many plant species subjected to excess salinity is often associated with a reduction in photosynthetic capacity (Belkhodja et al. 1999). The reduction in photosynthetic capacity due to salt stress is associated with reduced SC (Belkhodja et al. 1999). Measurement of cell membrane stability (CMS) has often been used for screening for drought and salinity tolerance in various plants such as sorghum, maize, rice and wheat (Jyothsnakumari et al. 2009). The decreased gibberellic acid (GA3) and increased abscisic acid (ABA) contents were reported in salt-stressed plants, which led to the suggestion that salt stress induces changes in RWC and membrane permeability (Kaya et al. 2009). Therefore, an alternative strategy to ameliorate salt stress could be exogenous application of plant growth regulators (Levent Tuna et al. 2008). So, focusing on using of phytohormones such as GA3 (which has important effects on regulation of plant reaction to environment and control of some induced genes in stress condition) is necessary (Nagvi 1999). It has been reported that GA3 treatment reduces the adverse effects of salt stress (Chakrabarti & Mukherji 2003). Phytohormones have also a control role in RWC and membrane permeability (Kaya et al. 2009). It was found that GA3 increases plant growth and pigment content of salinized plants (Aldesuquy & Gaber 1993). Prakash and Prathapasenan (1990) observed a significant decrease in the levels of gibberellins and cytokinins and an abrupt rise in ABA content in salt-stressed plants (Prakash & Prathapasenan 1990). ABA has an important function in the ability of rice cultivars to tolerate conditions of stress (Abdel-Haleem & Tanimoto 2008), so ABA spraying increases expression of numerous reactive genes in salt-stressed rice cultivars (Gupta et al. 1998). Wheat (Triticum aestivum L.) is one of the most important crops in Iran and throughout the world, which has a special importance in human nutrition (Esfandiari et al. 2007). Thus, this experiment was carried out to evaluate the effects of ABA and GA3 on some physiological traits of wheat cultivars including membrane stability (MS), chlorophyll content (CC), chlorophyll fluorescence (CF), stomatal conductance (SC), relative water content (RWC) under saline conditions in order to evaluate inhibitory or stimulatory effects and importance of the hormones on above-mentioned parameters in the three Iranian indigenous wheat cultivars in saline conditions. MATERIALS AND METHODS This study was carried out using a factorial experiment based on a completely randomized design with 4 replicates in a greenhouse at The University of Mohaghegh Ardabili, Ardabil, Iran. Treatments were salinity [Control : 0 (Ctrls), 3.5 and 7 dS/m, phytohormones (GA3 : 50 mg/l, ABA : 100 mg/l, and control (Ctrlh)], and 3 wheat (Triticum aestivum) cultivars [Gascogen (G), Zagros (Z) and Kuhdasht (K)]. The seeds were sterilized for 4 minutes in sodium hypochlorite 0.5%, rinsed in distilled water and were placed at 23 ± 1°C in a germinator. Then, germinated seeds were sown in pots (34 cm length, 25 cm width, and 18 cm height), which had sandy clay loam soil. Salt solutions (2.08 and 4.17 g/l NaCl, respectively) were poured on pots one week before planting, to reach 3.5 and 7 dS/m salinity levels. The initial salinity level (EC) of the soils in the pots was 3.93 dS/m. The phytohormones were foliarly sprayed in three stages, including tillering, shooting and flag leaf appearance stage (FLAS). Membrane Stability (MS) MS was measured once before hormone spraying and another time at FLAS. For MS determination, the procedures of Khandan Bejandi et al. (2009) that is briefly mentioned here was followed, the fully expanded leaves at each stage were selected and onecentimetre pieces were cut from the middle of the leaves. Then, 0.3 g of the leaf samples were weighed and rinsed in distilled water. These pieces were put in tubes with 25 ml distilled water and 25 ml polyethylene glycol 40% (PEG 6000) and then were put in an incubator for 24 hours (10°C). After that time, the solution of tubes was poured out, and the pieces were rinsed. Then, control and treatment samples were put in 25 ml distilled water for 24 hours. After a certain time, electrical conductivity of solutions was measured, and the samples were autoclaved for 15 min, cooled down to room temperature and then measured once more. MS is defined as Eq. (1) according to Khandan Bejandi et al. (2009): CMS [%] = 1 ­ (1 ­ T1 / T2) / (1 ­ C1 / C2) ×100 (1) Stomatal Conductance (SC) SC was measured after hormone spraying at FLAS by porometer (Decagon, USA). Relative Water Content (RWC) RWC was estimated according to Khandan Bejandi et al. (2009) using the following equation: RWC = (FW ­ DW) / (TW ­ DW) ×100 (2) where FW, DW and TW are the fresh, dry and turgid weight of leaves, respectively. Samples were taken from newly expanded leaves. Leaves were immediately weighed and then immersed in distilled water for 5 hours. Thereafter, turgid weight was obtained and finally, dry weight was measured 24 hours after being put at 75°C oven. Statistical Analysis After normalization test, the data were analyzed by SAS 9.1 and SPSS 16.0 software. Means were compared by Slice command in SAS at 1% statistical probability level. RESULTS AND DISCUSSION The results showed that interactions among cultivars, salinity, and hormones on the evaluated traits were significant (Table 1). Based on the interaction between hormones and salinity, GA3 decreased more inhibitory effects of salinity as compared to ABA (Table 2). Before the hormone spraying, the highest MS was achieved in K and Ctrls and the lowest value was obtained in G at 7 dS/m salinity level (Figure 1). After the hormone spraying, the highest MS (about 20%) was achieved in K, without salinity and under GA3 treatment and the lowest MS was observed in G, without salinity and under ABA treatment. By spraying of GA3, MS was increased under control (0) and both salinity levels (3.5 and 7 dS/m). Also, spraying of ABA in interaction with salinity levels increased MS as compared to Ctrls and Ctrlh, but this increase was less than the content resulted from spraying of GA3 (Table 2). Tellingly, GA3 as compared to ABA showed more MS. However, interestingly enough, GA3 treatment under Ctrls did not increase MS. By contrast, in G, GA3 treatment at 3.5 and 7 dS/m salinity levels, in In the above equation, C and T are the electrical conductivity for control and PEG treatment, respectively. Indices 1 and 2 are the first and second conductance, respectively. Chlorophyll Content (CC) CC was estimated at two stages, once before hormone spraying (4 leaves stage) and another time at FLAS by hand-held chlorophyll meter (SPAD-502, Minolta, Japan). Chlorophyll Fluorescence (CF) CF was measured after hormone spraying at FLAS by a chlorophyll fluorometer (Optiscience, USA). comparison to Ctrls caused more decrease in MS as compared to the same condition for ABA. In K like G and contrary to Z, ABA in 3.5 and 7 dS/m salinity as compared to Ctrls was better than GA3 treatment. Ashraf et al. (2005) stated that membrane lipids stability under saline condition seldom remains intact. A major impact of environmental stress on plants is cellular membrane modification due to salt stress, expressed in increased permeability and leakage of ions (Khandan Bejandi et al. 2009). Studies showed that abiotic stresses, including salinity generated reactive oxygen species and membrane lipid peroxidation in leaves and ears of wheat (Beltrano et al. 1997), caused decrease in MS. Khandan Bejandi et c d e f Membrane stability [%] Control 3.5 dS/m 7 dS/m Gascogen Zagros Kuhdasht Figure 1. Membrane stability [%] under salinity stress in the three wheat cultivars T a b l e 1 Analysis of variance for effects of cultivar, salinity and phytohormones on evaluated traits in wheat MS S.O.V Cultivar (C) Salinity (S) Hormone (H) C×S C×H H×S C×S×H Error CV [%] + and df 2 2 2 4 4 4 8 81 Membrane stability 2,638.021 123.486 109.446 15.156 Chlorophyll content 862.156 162.443 58.609 Stomatal conductance 576.411 1,518.478 1,789.296 215.310 Chlorophyll fluorescence 0.001 0.001 0.035 0.002 Relative water content 2,839.828 594.008 816.332 246.715 23.098 34.226 22.056 0.333 1.28 39.406 12.108 0.191 851.088 0.001 18.542 13.668 0.062 0.074ns 0.192 0.060 142.363 40.494 0.066 0.001 0.0001ns 0.0001 0.32 0.55 0.53 0.88 ns : Significant at 5% and 1% of probability levels, respectively; : Non-significant al. (2009) stated that the accumulation of reactive oxygen species under stress conditions may damage many cell components, such as lipids, proteins, carbohydrates and nucleic acids as a result of cell membrane lipids peroxidation. The excess of NaCl leads to the loss of potassium due to membrane depolarization by sodium ions (Turan et al. 2009). Bhutta investigation (2011) showed that MS increased under the non-saline environment in the leaves of wheat seedlings, in so far as under low saline levels, MS decreased 22% and 29% after 20 and 40 days treatments, respectively. MS showed more decrease (27%) under high saline levels (160 nM NaCl) after 40 days. In other words, MS in wheat leaves reached the lowest levels in both wheat genotypes (S24 and DN-27). T a b l e 2 Mean comparison of interaction among variety × salinity × hormone with evaluated traits in wheat Treatment H1 G H2 H3 H1 S1 Z H2 H3 H1 K H2 H3 H1 G H2 H3 S2 H1 Z H2 H3 H1 K H2 H3 H1 G H2 H3 S3 H1 Z H2 H3 H1 K H2 Membrane stability [%] 85.65b 93.79 75.62 74.06 Chlorophyll content 53.79b 52.03 42.90 43.85 Stomatal conduct [mmol/m2/s] 40.57e 33.43 23.47 41.00 Chlorophyll fluorescence 0.7100b 0.6733 0.7100 0.6833 Relative water content 84.97c 83.50d 87.49b 54.97h 50.77i 57.55g 78.94e 69.66f 90.65a 87.60b 77.78c 91.55a 67.06f 63.61g 70.50e 67.11f 63.77g 73.33d 78.75b 67.67d 82.73a 48.22h 42.83i 63.45e 34.97f 52.72g 74.37e 54.67a 60.98a 0.6900c 77.00c 41.04f 16.25i 0.6000g 62.68g 63.65f 59.26 86.84 84.42 82.99 64.43 87.42 46.79h 45.33i 48.62 47.08 38.87 39.84 31.80g 46.66c 52.40 0.7267a 0.6100f 0.6700 85.94b 48.19b 77.34b 46.08 56.46 60.25 0.6800c 0.6433 0.6933 0.6767 0.6233 0.7100 84.01c 49.88a 79.88a 0.6567e 85.60b 37.07i 51.31e 0.6367g 63.68f 43.79e 42.14 50.91 35.96h 31.30 55.45 0.7067a 61.46g 45.62d 37.97g 0.6833c 88.60a 85.26d 86.10 83.99 65.54 49.09c 51.77a 37.58 39.15 36.08i 70.01a 53.79 64.96 48.17 0.6500f 0.6567e 0.6867 0.6667 0.6333 87.27b 36.01i 46.19h 0.6100h 64.16f 41.85e 40.05 58.41d 0.7000b H3 63.31h 42.89d 66.96b 0.6667d 73.22c Similar letters in each column are not significant at 1% and 5% probability level S1 = Control, S2 = 3.5 dS/m salinity, S3 = 7 dS/m salinity G = Gascogen cultivar, K = Kuhdasht cultivar, Z = Zagros cultivar, H1 = control, H2 = Abscisic acid, H3 = Gibberellic acid Agriculture (Ponohospodárstvo), 59, 2013 (1): 33­41 Before hormone spraying, with increase in salinity, significant reduction in CC was observed and the highest reduction reached 25.06% in Z at 3.5 dS/m salinity (Figure 2). After hormone spraying, the highest CC was obtained under GA3 treatment (Table 2). It seems that GA3 has an important effect on Chl stability and synthesis, even under salinity condition. Reduction in leaf CC under NaCl stress is attributed to the destruction of Chl pigments and the instability of the pigment protein complex (Levitt 1980). The Chl depletion may be a result of inhibition of Chl biosynthesis. This inhibition is due to an increase in ethylene production by the elevated NaCl content (Khan 2003). Reduction in Chl concentrations is probably because of the inhibitory effect of the accumulated ions of various salts on the biosynthesis of different Chl fractions. Also, decrease in CC may be based on the formation of proteolytic enzymes such as chlorophyllase, which is responsible for the Chl degradation (Levent Tuna et al. 2008). It has also been shown that GA3 alleviates the effects of salt stress on pigments (Shah 2007). This may well be attributed to the GA3-generated enhancement of ultra-structural morphogenesis of plastids coupled with retention of Chl and delay of senescence caused by the hormone treatment (Shah 2007). Decrease in CC under salinity stress can be for this reason which Chl degradation is more than Chl synthesis (Uzma & Asghari 2006). Sairam et al. (2002) reported that reduction of CC in a tolerant wheat variety was lower than in a sensitive one. Akbari Ghogdi et al. (2012) documented that salt treatment reduced the CC at tillering stage and decreased it at flowering stage in wheat cultivars. In the present experiment, ABA caused the lowest CF in salinity levels. However, with scrutiny comparison of GA3 and ABA, it can be understood that ABA in higher salinity levels caused much less decrement. The ratio Fv/Fm is proportional in order to show maximum amount of PSII. The ratio for a functional leaf varies between 0.75 and 0.85 and a decline in this ratio is an indicator for photoinhibitory damage (DeEll et al. 1999). There was a significant increase in SC with spraying of GA3 under saline condition in comparison to the Ctrls condition. The lowest amount of SC was achieved in Z under ABA treatment and the Ctrls (Table 2). Interaction between ABA and salinity probably decreases SC to the lower amount than that of control. In brief, in the three cultivars G, K and Z and salinity levels, ABA as compared a d b g i c f h Chlorophyll content Control 3.5 dS/m 7 dS/m Gascogen Kuhdasht Zagros Figure 2. Chlorophyll content under salinity stress in the three wheat cultivars to GA3, decreased SC. Environmental factors such as salinity that affect water quality lead to changes in stomatal opening (Nelson et al. 1998). Salinity increases stomatal resistance, which could be explained by inhibition of plant growth due to water stress (Chatrath et al. 2000). To control the water status of stomatal pore, plants may use two hormone mediated strategies: 1) is to control stomatal pore width, and 2) is to control the hydraulic conductivity of the root (Ludewig et al. 1988). ABA in guard cells activates ionic channels and proton pump that are associated with stomatal closure (Goh et al. 2009). H2O2 acts as a local or systemic signal for leaf stomata closure (Chaves et al. 2009). Decrease in SC indicates that changes in the osmotic situation of root medium quickly affect shoot water relations (Rodriguez et al. 1997). Salinity causes ABA production in roots which is translocated to shoot and induces stomatal closure and limitation of cell growth (Lobna et al. 2009). Rahnama et al. (2010) reported that salinity reduced SC in different wheat genotypes and the largest reduction was observed in sensitive genotypes of wheat. Also, they suggested that root signals presumably cause a large decrease in SC of wheat genotypes under salinity. ABA in Ctrls reduced RWC. However, interaction between GA3 and salinity levels increased the content of RWC (Table 2). RWC decreased by increase in salinity levels in wheat genotypes (Akbari Ghogdi et al. 2012). It seems that decrease in RWC decreases the turgor due to water limitation. Such an increase in the RWC of salinized plants over that of the control or hormone-treated salinized plants may be due to the accumulation of ABA to levels that cause stomatal closure, which reduces water loss, maintains turgor and improves the water use efficiency of plants and allows more growth with a given supply of water (Aldesuquy & Ibrahim 2001). Generally, it is expected that GA3 moderates the deleterious condition of saline stress in comparison to ABA in wheat. It is known that conduction of the present study in field is essential in order to assess effects of the factors in uncontrollable condition. However, in natural condition there are many factors that are out of control, thus the results of in natura studies may deviate from those of greenhouse condition. It is recommended to evaluate the factors of the present greenhouse study in field. CONCLUSION According to the present experiment, hormones and high saline levels have different effects on various parameters in wheat cultivars. High saline levels cause higher chlorophyll fluorescence, and relative water content. In contrast, lower membrane stability, chlorophyll content, and stomatal conductance have been caused under saline condition. Abscisic acid is more effective for decreasing adverse consequences of salinity on chlorophyll fluorescence, membrane stability and, relative water content. However, gibberellic acid has beneficial influence on chlorophyll content and stomatal conductance as compared by abscisic acid under saline stress. In sum, phytohormones play a significant role in moderating the effects of salinity on photosynthetic traits and membrane stability. Acknowledgments. The authors thank Prof. M. Pessarakli, Research Professor (School of Plant Sciences, College of Agriculture and Life Sciences, The University of Arizona, Tucson, Arizona, USA) for helpful discussions and advice. This research was supported by an MSc grant from The University of Mohaghegh Ardabili, Iran, Ardabil. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Agriculture de Gruyter

Interaction of Salinity and Phytohormones on Wheat Photosynthetic Traits and Membrane Stability

Agriculture , Volume 59 (1) – Mar 1, 2013

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DOI: 10.2478/agri-2013-0004 ARMAN PAZUKI 1, MOHAMMAD SEDGHI 2, FATEMEH AFLAKI 2* University of Guilan, Rasht University of Mohaghegh Ardabili, Ardabil PAZUKI, A. ­ SEDGHI, M. ­ AFLAKI, F.: Interaction of salinity and phytohormones on wheat photosynthetic traits and membrane stability. Agriculture (Ponohospodárstvo), vol. 59, 2013, no. 1, pp. 33­41. To evaluate phytohormones effects on stomatal conductance, chlorophyll fluorescence, membrane stability, relative water content and chlorophyll content under salinity, a factorial experiment with 4 replicates was conducted. Treatments were salinity (0, 3.5 and 7 dS/m), phytohormones (control, gibberellic acid and abscisic acid) and wheat cultivars (Gascogen, Zagros, and Kuhdasht). Results showed that a high level of salinity increased chlorophyll fluorescence and relative water content, while membrane stability, chlorophyll content, and stomatal conductance were decreased. Abscisic acid treatment had more effective role in membrane stability. Although membrane stability was much more under gibberellic acid treatment, restoration of membrane stability was considerable under abscisic acid treatment for Gascogen and Kuhdasht cultivars. Spraying of gibberellic acid induced the highest chlorophyll content in the three salinity levels and all of the cultivars. The maximum amount of stomatal conductance was achieved under gibberellic acid treatment. Abscisic acid caused less chlorophyll fluorescence in comparison to gibberellic acid. About relative water content, abscisic acid was effective in high salinity levels so that it caused stomatal closure, which reduced water loss and maintained turgor in plants. Key words: Triticum aestivum, NaCl, gibberellic acid, abscisic acid, chlorophyll fluorescence, stomatal conductance Salinity is one of the major abiotic stresses affecting plant growth, development and productivity (Rahnama & Ebrahimzadeh 2004). Salinity is a complex environmental constraint that presents two main components: i) osmotic component due to decrease in the external osmotic potential of the soil solution and ii) ionic component linked to the accumulation of ions which become toxic at high concentrations (mainly Na + and Cl - ) (El-Bassiouny & Bekheta 2004). It is estimated that over 800 million hectares of land in the world are affected by both salinity and sodicity (Munns 2005). Plants exposed to salt stress must undergo changes in their metabolism in order to survive in the deleterious condition of the stress (Rahnama & Ebrahimzadeh 2004). These changes include stomatal conductance (SC), photosynthetic efficiency, water and nutrients availability (Munns & Termaat 1986). Chlorophyll content (CC), relative water content (RWC) and SC are affected by increasing salinity (Adnan Shahid et al. 2008). RWC represents a useful indicator of the state of plant water balance, because it expresses the absolute amount of water, which plant requires reaching full saturation (González & González-Vilar 2001). Leaf RWC is a useful trait for selection of tolerant plants in saline condition (Schonfeld et al. 1988) and is significantly reduced under salinity stress (El-Tayeb 2005). Furthermore, salinity stress also reduces RWC in sensitive wheat cultivars seedlings (Aldesuquy & Gaber 1993). Fatemeh Aflaki* (Corresponding Author), Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Mohaghegh Ardabili, Ardabil, Iran. E-mail: fa_aflaki@yahoo.com Chlorophyll (Chl) is one of the most important pigments, and is responsible for green colour in plants. Changes in photosynthetic parameters could potentially be used as a screening method for salinity tolerance in plants, because more tolerant cultivars are expected to exhibit less disturbances in photosynthetic processes (Belkhodja et al. 1999). Generally, Chl content is reduced under salinity conditions (Iqbal et al. 2006). One of the leading parameters for the estimation of salinity stress effects is chlorophyll fluorescence (CF). CF is a subtle reflection of primary reactions of photosynthesis (Sayed 2003). The ratio of Fv/Fm is proportional in order to show maximum amount of photosystem II (PSII), and this ratio is sensitive to environmental changes (Sayed 2003). Krishnaraj et al. (1993) used measurement of CF for screening of tolerant wheat genotypes (Krishnaraj et al. 1993). Various stress conditions may reduce the rate of photosynthesis, and disturb the light-driven photosynthetic electron transport via CF fluorescence (Allahverdiev et al. 1998). The inverse relationship between in vivo CF and photosynthetic activity can be used to study the potential photosynthetic activity of leaves and detect stress effects on green plants. There are several parameters of the in vivo CF which can be applied for detecting stress and damages to the photosynthetic apparatus. Variable fluorescence (Fv) and initial fluorescence (Fo) ratio are two of these parameters, and they show the efficiency of photosynthesis (Allahverdiev et al. 1998). The decline in productivity in many plant species subjected to excess salinity is often associated with a reduction in photosynthetic capacity (Belkhodja et al. 1999). The reduction in photosynthetic capacity due to salt stress is associated with reduced SC (Belkhodja et al. 1999). Measurement of cell membrane stability (CMS) has often been used for screening for drought and salinity tolerance in various plants such as sorghum, maize, rice and wheat (Jyothsnakumari et al. 2009). The decreased gibberellic acid (GA3) and increased abscisic acid (ABA) contents were reported in salt-stressed plants, which led to the suggestion that salt stress induces changes in RWC and membrane permeability (Kaya et al. 2009). Therefore, an alternative strategy to ameliorate salt stress could be exogenous application of plant growth regulators (Levent Tuna et al. 2008). So, focusing on using of phytohormones such as GA3 (which has important effects on regulation of plant reaction to environment and control of some induced genes in stress condition) is necessary (Nagvi 1999). It has been reported that GA3 treatment reduces the adverse effects of salt stress (Chakrabarti & Mukherji 2003). Phytohormones have also a control role in RWC and membrane permeability (Kaya et al. 2009). It was found that GA3 increases plant growth and pigment content of salinized plants (Aldesuquy & Gaber 1993). Prakash and Prathapasenan (1990) observed a significant decrease in the levels of gibberellins and cytokinins and an abrupt rise in ABA content in salt-stressed plants (Prakash & Prathapasenan 1990). ABA has an important function in the ability of rice cultivars to tolerate conditions of stress (Abdel-Haleem & Tanimoto 2008), so ABA spraying increases expression of numerous reactive genes in salt-stressed rice cultivars (Gupta et al. 1998). Wheat (Triticum aestivum L.) is one of the most important crops in Iran and throughout the world, which has a special importance in human nutrition (Esfandiari et al. 2007). Thus, this experiment was carried out to evaluate the effects of ABA and GA3 on some physiological traits of wheat cultivars including membrane stability (MS), chlorophyll content (CC), chlorophyll fluorescence (CF), stomatal conductance (SC), relative water content (RWC) under saline conditions in order to evaluate inhibitory or stimulatory effects and importance of the hormones on above-mentioned parameters in the three Iranian indigenous wheat cultivars in saline conditions. MATERIALS AND METHODS This study was carried out using a factorial experiment based on a completely randomized design with 4 replicates in a greenhouse at The University of Mohaghegh Ardabili, Ardabil, Iran. Treatments were salinity [Control : 0 (Ctrls), 3.5 and 7 dS/m, phytohormones (GA3 : 50 mg/l, ABA : 100 mg/l, and control (Ctrlh)], and 3 wheat (Triticum aestivum) cultivars [Gascogen (G), Zagros (Z) and Kuhdasht (K)]. The seeds were sterilized for 4 minutes in sodium hypochlorite 0.5%, rinsed in distilled water and were placed at 23 ± 1°C in a germinator. Then, germinated seeds were sown in pots (34 cm length, 25 cm width, and 18 cm height), which had sandy clay loam soil. Salt solutions (2.08 and 4.17 g/l NaCl, respectively) were poured on pots one week before planting, to reach 3.5 and 7 dS/m salinity levels. The initial salinity level (EC) of the soils in the pots was 3.93 dS/m. The phytohormones were foliarly sprayed in three stages, including tillering, shooting and flag leaf appearance stage (FLAS). Membrane Stability (MS) MS was measured once before hormone spraying and another time at FLAS. For MS determination, the procedures of Khandan Bejandi et al. (2009) that is briefly mentioned here was followed, the fully expanded leaves at each stage were selected and onecentimetre pieces were cut from the middle of the leaves. Then, 0.3 g of the leaf samples were weighed and rinsed in distilled water. These pieces were put in tubes with 25 ml distilled water and 25 ml polyethylene glycol 40% (PEG 6000) and then were put in an incubator for 24 hours (10°C). After that time, the solution of tubes was poured out, and the pieces were rinsed. Then, control and treatment samples were put in 25 ml distilled water for 24 hours. After a certain time, electrical conductivity of solutions was measured, and the samples were autoclaved for 15 min, cooled down to room temperature and then measured once more. MS is defined as Eq. (1) according to Khandan Bejandi et al. (2009): CMS [%] = 1 ­ (1 ­ T1 / T2) / (1 ­ C1 / C2) ×100 (1) Stomatal Conductance (SC) SC was measured after hormone spraying at FLAS by porometer (Decagon, USA). Relative Water Content (RWC) RWC was estimated according to Khandan Bejandi et al. (2009) using the following equation: RWC = (FW ­ DW) / (TW ­ DW) ×100 (2) where FW, DW and TW are the fresh, dry and turgid weight of leaves, respectively. Samples were taken from newly expanded leaves. Leaves were immediately weighed and then immersed in distilled water for 5 hours. Thereafter, turgid weight was obtained and finally, dry weight was measured 24 hours after being put at 75°C oven. Statistical Analysis After normalization test, the data were analyzed by SAS 9.1 and SPSS 16.0 software. Means were compared by Slice command in SAS at 1% statistical probability level. RESULTS AND DISCUSSION The results showed that interactions among cultivars, salinity, and hormones on the evaluated traits were significant (Table 1). Based on the interaction between hormones and salinity, GA3 decreased more inhibitory effects of salinity as compared to ABA (Table 2). Before the hormone spraying, the highest MS was achieved in K and Ctrls and the lowest value was obtained in G at 7 dS/m salinity level (Figure 1). After the hormone spraying, the highest MS (about 20%) was achieved in K, without salinity and under GA3 treatment and the lowest MS was observed in G, without salinity and under ABA treatment. By spraying of GA3, MS was increased under control (0) and both salinity levels (3.5 and 7 dS/m). Also, spraying of ABA in interaction with salinity levels increased MS as compared to Ctrls and Ctrlh, but this increase was less than the content resulted from spraying of GA3 (Table 2). Tellingly, GA3 as compared to ABA showed more MS. However, interestingly enough, GA3 treatment under Ctrls did not increase MS. By contrast, in G, GA3 treatment at 3.5 and 7 dS/m salinity levels, in In the above equation, C and T are the electrical conductivity for control and PEG treatment, respectively. Indices 1 and 2 are the first and second conductance, respectively. Chlorophyll Content (CC) CC was estimated at two stages, once before hormone spraying (4 leaves stage) and another time at FLAS by hand-held chlorophyll meter (SPAD-502, Minolta, Japan). Chlorophyll Fluorescence (CF) CF was measured after hormone spraying at FLAS by a chlorophyll fluorometer (Optiscience, USA). comparison to Ctrls caused more decrease in MS as compared to the same condition for ABA. In K like G and contrary to Z, ABA in 3.5 and 7 dS/m salinity as compared to Ctrls was better than GA3 treatment. Ashraf et al. (2005) stated that membrane lipids stability under saline condition seldom remains intact. A major impact of environmental stress on plants is cellular membrane modification due to salt stress, expressed in increased permeability and leakage of ions (Khandan Bejandi et al. 2009). Studies showed that abiotic stresses, including salinity generated reactive oxygen species and membrane lipid peroxidation in leaves and ears of wheat (Beltrano et al. 1997), caused decrease in MS. Khandan Bejandi et c d e f Membrane stability [%] Control 3.5 dS/m 7 dS/m Gascogen Zagros Kuhdasht Figure 1. Membrane stability [%] under salinity stress in the three wheat cultivars T a b l e 1 Analysis of variance for effects of cultivar, salinity and phytohormones on evaluated traits in wheat MS S.O.V Cultivar (C) Salinity (S) Hormone (H) C×S C×H H×S C×S×H Error CV [%] + and df 2 2 2 4 4 4 8 81 Membrane stability 2,638.021 123.486 109.446 15.156 Chlorophyll content 862.156 162.443 58.609 Stomatal conductance 576.411 1,518.478 1,789.296 215.310 Chlorophyll fluorescence 0.001 0.001 0.035 0.002 Relative water content 2,839.828 594.008 816.332 246.715 23.098 34.226 22.056 0.333 1.28 39.406 12.108 0.191 851.088 0.001 18.542 13.668 0.062 0.074ns 0.192 0.060 142.363 40.494 0.066 0.001 0.0001ns 0.0001 0.32 0.55 0.53 0.88 ns : Significant at 5% and 1% of probability levels, respectively; : Non-significant al. (2009) stated that the accumulation of reactive oxygen species under stress conditions may damage many cell components, such as lipids, proteins, carbohydrates and nucleic acids as a result of cell membrane lipids peroxidation. The excess of NaCl leads to the loss of potassium due to membrane depolarization by sodium ions (Turan et al. 2009). Bhutta investigation (2011) showed that MS increased under the non-saline environment in the leaves of wheat seedlings, in so far as under low saline levels, MS decreased 22% and 29% after 20 and 40 days treatments, respectively. MS showed more decrease (27%) under high saline levels (160 nM NaCl) after 40 days. In other words, MS in wheat leaves reached the lowest levels in both wheat genotypes (S24 and DN-27). T a b l e 2 Mean comparison of interaction among variety × salinity × hormone with evaluated traits in wheat Treatment H1 G H2 H3 H1 S1 Z H2 H3 H1 K H2 H3 H1 G H2 H3 S2 H1 Z H2 H3 H1 K H2 H3 H1 G H2 H3 S3 H1 Z H2 H3 H1 K H2 Membrane stability [%] 85.65b 93.79 75.62 74.06 Chlorophyll content 53.79b 52.03 42.90 43.85 Stomatal conduct [mmol/m2/s] 40.57e 33.43 23.47 41.00 Chlorophyll fluorescence 0.7100b 0.6733 0.7100 0.6833 Relative water content 84.97c 83.50d 87.49b 54.97h 50.77i 57.55g 78.94e 69.66f 90.65a 87.60b 77.78c 91.55a 67.06f 63.61g 70.50e 67.11f 63.77g 73.33d 78.75b 67.67d 82.73a 48.22h 42.83i 63.45e 34.97f 52.72g 74.37e 54.67a 60.98a 0.6900c 77.00c 41.04f 16.25i 0.6000g 62.68g 63.65f 59.26 86.84 84.42 82.99 64.43 87.42 46.79h 45.33i 48.62 47.08 38.87 39.84 31.80g 46.66c 52.40 0.7267a 0.6100f 0.6700 85.94b 48.19b 77.34b 46.08 56.46 60.25 0.6800c 0.6433 0.6933 0.6767 0.6233 0.7100 84.01c 49.88a 79.88a 0.6567e 85.60b 37.07i 51.31e 0.6367g 63.68f 43.79e 42.14 50.91 35.96h 31.30 55.45 0.7067a 61.46g 45.62d 37.97g 0.6833c 88.60a 85.26d 86.10 83.99 65.54 49.09c 51.77a 37.58 39.15 36.08i 70.01a 53.79 64.96 48.17 0.6500f 0.6567e 0.6867 0.6667 0.6333 87.27b 36.01i 46.19h 0.6100h 64.16f 41.85e 40.05 58.41d 0.7000b H3 63.31h 42.89d 66.96b 0.6667d 73.22c Similar letters in each column are not significant at 1% and 5% probability level S1 = Control, S2 = 3.5 dS/m salinity, S3 = 7 dS/m salinity G = Gascogen cultivar, K = Kuhdasht cultivar, Z = Zagros cultivar, H1 = control, H2 = Abscisic acid, H3 = Gibberellic acid Agriculture (Ponohospodárstvo), 59, 2013 (1): 33­41 Before hormone spraying, with increase in salinity, significant reduction in CC was observed and the highest reduction reached 25.06% in Z at 3.5 dS/m salinity (Figure 2). After hormone spraying, the highest CC was obtained under GA3 treatment (Table 2). It seems that GA3 has an important effect on Chl stability and synthesis, even under salinity condition. Reduction in leaf CC under NaCl stress is attributed to the destruction of Chl pigments and the instability of the pigment protein complex (Levitt 1980). The Chl depletion may be a result of inhibition of Chl biosynthesis. This inhibition is due to an increase in ethylene production by the elevated NaCl content (Khan 2003). Reduction in Chl concentrations is probably because of the inhibitory effect of the accumulated ions of various salts on the biosynthesis of different Chl fractions. Also, decrease in CC may be based on the formation of proteolytic enzymes such as chlorophyllase, which is responsible for the Chl degradation (Levent Tuna et al. 2008). It has also been shown that GA3 alleviates the effects of salt stress on pigments (Shah 2007). This may well be attributed to the GA3-generated enhancement of ultra-structural morphogenesis of plastids coupled with retention of Chl and delay of senescence caused by the hormone treatment (Shah 2007). Decrease in CC under salinity stress can be for this reason which Chl degradation is more than Chl synthesis (Uzma & Asghari 2006). Sairam et al. (2002) reported that reduction of CC in a tolerant wheat variety was lower than in a sensitive one. Akbari Ghogdi et al. (2012) documented that salt treatment reduced the CC at tillering stage and decreased it at flowering stage in wheat cultivars. In the present experiment, ABA caused the lowest CF in salinity levels. However, with scrutiny comparison of GA3 and ABA, it can be understood that ABA in higher salinity levels caused much less decrement. The ratio Fv/Fm is proportional in order to show maximum amount of PSII. The ratio for a functional leaf varies between 0.75 and 0.85 and a decline in this ratio is an indicator for photoinhibitory damage (DeEll et al. 1999). There was a significant increase in SC with spraying of GA3 under saline condition in comparison to the Ctrls condition. The lowest amount of SC was achieved in Z under ABA treatment and the Ctrls (Table 2). Interaction between ABA and salinity probably decreases SC to the lower amount than that of control. In brief, in the three cultivars G, K and Z and salinity levels, ABA as compared a d b g i c f h Chlorophyll content Control 3.5 dS/m 7 dS/m Gascogen Kuhdasht Zagros Figure 2. Chlorophyll content under salinity stress in the three wheat cultivars to GA3, decreased SC. Environmental factors such as salinity that affect water quality lead to changes in stomatal opening (Nelson et al. 1998). Salinity increases stomatal resistance, which could be explained by inhibition of plant growth due to water stress (Chatrath et al. 2000). To control the water status of stomatal pore, plants may use two hormone mediated strategies: 1) is to control stomatal pore width, and 2) is to control the hydraulic conductivity of the root (Ludewig et al. 1988). ABA in guard cells activates ionic channels and proton pump that are associated with stomatal closure (Goh et al. 2009). H2O2 acts as a local or systemic signal for leaf stomata closure (Chaves et al. 2009). Decrease in SC indicates that changes in the osmotic situation of root medium quickly affect shoot water relations (Rodriguez et al. 1997). Salinity causes ABA production in roots which is translocated to shoot and induces stomatal closure and limitation of cell growth (Lobna et al. 2009). Rahnama et al. (2010) reported that salinity reduced SC in different wheat genotypes and the largest reduction was observed in sensitive genotypes of wheat. Also, they suggested that root signals presumably cause a large decrease in SC of wheat genotypes under salinity. ABA in Ctrls reduced RWC. However, interaction between GA3 and salinity levels increased the content of RWC (Table 2). RWC decreased by increase in salinity levels in wheat genotypes (Akbari Ghogdi et al. 2012). It seems that decrease in RWC decreases the turgor due to water limitation. Such an increase in the RWC of salinized plants over that of the control or hormone-treated salinized plants may be due to the accumulation of ABA to levels that cause stomatal closure, which reduces water loss, maintains turgor and improves the water use efficiency of plants and allows more growth with a given supply of water (Aldesuquy & Ibrahim 2001). Generally, it is expected that GA3 moderates the deleterious condition of saline stress in comparison to ABA in wheat. It is known that conduction of the present study in field is essential in order to assess effects of the factors in uncontrollable condition. However, in natural condition there are many factors that are out of control, thus the results of in natura studies may deviate from those of greenhouse condition. It is recommended to evaluate the factors of the present greenhouse study in field. CONCLUSION According to the present experiment, hormones and high saline levels have different effects on various parameters in wheat cultivars. High saline levels cause higher chlorophyll fluorescence, and relative water content. In contrast, lower membrane stability, chlorophyll content, and stomatal conductance have been caused under saline condition. Abscisic acid is more effective for decreasing adverse consequences of salinity on chlorophyll fluorescence, membrane stability and, relative water content. However, gibberellic acid has beneficial influence on chlorophyll content and stomatal conductance as compared by abscisic acid under saline stress. In sum, phytohormones play a significant role in moderating the effects of salinity on photosynthetic traits and membrane stability. Acknowledgments. The authors thank Prof. M. Pessarakli, Research Professor (School of Plant Sciences, College of Agriculture and Life Sciences, The University of Arizona, Tucson, Arizona, USA) for helpful discussions and advice. This research was supported by an MSc grant from The University of Mohaghegh Ardabili, Iran, Ardabil.

Journal

Agriculturede Gruyter

Published: Mar 1, 2013

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