Effect of Salinity Stress and Salicylic Acid on Morpho-physiological and Growth Characteristics Satureja mutica Fisch. & C. A. Mey.
Borzou Yousefi
1
(
1. PHD candidate of Plant Physiology, Department of Biology, Faculty of Science, Bu-Ali Sina university, Hamadan, Iran.
2. Department of Natural resources, Kermanshah Agricultural and Natural Resources Research and Education Center,
)
Roya Karamian
2
(
Prof of Plant Physiology, Department of Biology, Faculty of Science, Bu-Ali Sina university, Hamadan
)
Keywords:
Abstract :
Effect of salinity stress and salicylic acid on morpho-physiological and growth characteristics of forest savory (Satureja mutica Fisch. & C. A. Mey).
Borzou YousefiA* and Roya KaramianB
A PhD candidate, Dept. Biology, Faculty of Science, Bu-Ali Sina university, Hamadan, Iran.
*(Corresponding Author), Email: borzooyoosefi@yahoo.com,
B Prof. Dept. Biology, Faculty of Science, Bu-Ali Sina university, Hamadan, Iran.
Abstract:
Forest savory or white savory is a wild and perenial species which grows in the northwestern, north, and northeastern of Iran, and it is used in health industries and food products. In order to Investigating the moderating effect of salicylic acid on morpho-physiological traits of the forest savory under salinity stress conditions a pot experiment was performed as a factorial experiment based on a Completely Randomized Design (CRD) and three replications at the Kermanshah Agricultural & Natural Resources Research & Education Center, Iran, in 2019. Factor A was NaCl in four levels (0, 50, 100, and 150 mM) and Factor B was salicylic acid in two levels (0 and 2 mM). The results obtained from the analysis of variance showed a significant difference in the studied traits. The salinity stress reduced plant height, leaf area, leaf fresh and dry weight, root fresh and dry weight, shoot fresh and dry weight, relative water content, chlorophyll index, and maximum quantum yield of photosystem II (Fv/Fm value), but increased leaf electrical conductivity, and proline content. Soluble protein increased at 50 and 100 mM NaCl, but it significantly decreased at 150 mM NaCl. Salicylic acid caused an increase in plant height (17.54%), leaf area (24.62%), leaf fresh weight (12.41%), leaf dry weight (8.23%), shoot fresh weight (25.87%), shoot dry weight (13.75%), root fresh weight (70.99%), root dry weight (72.38%), relative water content (32.85%), maximum quantum yield of PSП (10.42%), and chlorophyll index SPAD (11.36%) compared to non-SA-treated plants. SA reduced proline content (12.07%) and leaf electrical conductivity (2.37%) in the SA-treated plants compared to non-SA-treated plants. The tolerance threshold for salinity of forest savory is less than 100 mm, and in soils more salty than this value, its growth and yield significantly decreases. Foliar spraying with 2 mM salicylic acid in forest savory plants exposed to salt stress, in the early stages of vegetative growth, enhanced the plant's tolerance to salinity and increased plant yield.
Keywords: Chlorophyll index, Photosynthesis, Proline, Maximum quantum yield of photosystem II, Salt stress, Savory
Introduction
Salinity decreases leaf water potential, disturbs ion homeostasis, and enhances reactive oxygen species (ROS). Consequently, salinity leads to osmotic and ionic stress (Arif et al., 2020), and salinity-induced oxidative stress that reduces the production of plant dry matter (Mushtaq et al., 2022). Under salinity stress, the osmotic potential of plant cells becomes more negative due to the presence of a high salt concentration in the soil, which creates an osmotic gradient which reduces water absorption to the plant and decreases turgor pressure (Betzen et al., 2019). Leaf relative water content (RWC) is an important indicator of water status in plants. In many plants, a decrease in the relative water content of leaves under different salt concentrations has been reported (Menezes et al., 2017, Arvin and Firuzeh, 2021).
Salinity can reduce the photosynthetic pigments, degradation of chlorophyll, decrease in net photosynthesis, and stunted growth (Mahajan and Pal, 2023). The maximum quantum yield of photosystem II (Fv/Fm) and chlorophyll index (SPAD) are useful variables to evaluate photosynthetic activities under stress conditions. Fv/Fm value is a sensitive indicator for the early detection of plant responses to environmental stresses (Bertamini et al., 2019), that provides valuable information for evaluating plant physiological changes under drought stress. Also, chlorophyll index (SPAD) can provide key information regarding photosynthesis and stress resistance (Yang et al., 2023). Fv/Fm shows the electron transfer capacity of photosystem II and also exhibits the quantum efficiency of pure photosynthesis. Photosynthetic performance, Fv/Fm, and SPAD are usually reduced under stress conditions (Wang et al., 2022).
Under osmotic stress, the accumulation of organic osmolytes such as carbohydrates, protein, and proline regulates the osmotic pressure of the cell (Behzadi Rad et al., 2021). Proline prevents electrolyte leakage, stabilizes protein structure and activity, regulates reactive oxygen species concentration, protects membrane integrity and stabilizes the subcellular structure in stressed plants (Zuo et al., 2022). Accumulation of proline under salt stress conditions has been reported in many plants such amaranth (Menezes et al., 2017). Several proteins accumulate in plants in response to salinity stress. Plant tissues normally respond to salt stress by degrading proteins or producing abundant salt stress related proteins (Athar et al., 2022). Severe stress causes the production of ROSs which leads to destruction of some enzymatic and structural proteins (Wang et al., 2015). Therefore, the changes in leaf soluble protein are a proper indicator for investigating the intensity of salt stress on plants.
Salicylic acid (SA) is one of the plant growth regulators (PGRs) that regulate plant growth and development by triggering many physiological and metabolic processes (Kaya et al., 2023).
Satureja mutica Fisch. & C. A. Mey is a native plant in the Caucasus region, Central Asia and Iran (Karimi et al., 2021) which is distributed in the pastures of the north, northeast, and northwest of Iran. Forest savory is used in traditional medicine to treat rheumatic pain, migraine, toothache and diarrhea. The phenolic monoterpenes are widely used constituents in pharmaceuticals, cosmetics, and food products (Nieto, 2020). Satureja is an important commercial source of phenolic monoterpenes (Stahl-Biskup, 2002). Thymol and carvacrol are best known phenolic monoterpenes. These aroma compounds are employed in medicine for their antibacterial, anti-spasmodic, antioxidant, and anti-cancer properties (Krause et al., 2021). They also serve as additives to cosmetics and are used in aromatherapy, because of their spicy, warm, and aromatic odors (Krause et al., 2021).Recently, the essential oil of this plant has been used as a food additive in animal feed (Imani M. et al., 2021 ). Domesticating of S. mutica (Fig. 1) has attracted the attention of medicinal plants researchers in recent years. The aim of this research was to investigate the ameliorating role of salicylic acid in the some morphological, physiological, and photosynthetic traits of S. mutica exposed to salt stress.
Fig. 1. Fv/Fm measurement by chlorophyll fluorimeter (a), Forest savory (S. mutica) plant (b), root of forest savory exposed to 2 mM SA+ 50 mM NaCl (c), and SPAD measurement by chlorophyll meter (d)
Materials and Methods
The seeds were collected in North Khorasan province, Iran (37°04" E; 57°29" N; 1610 m altitude; average rainfall was 347.7 mm; average annual temperature was 13.3 º C).
Research Method
A factorial experiment based on a Completely Randomized Design (CRD) with four levels of salinity (0-50-100-150 mM) and 2 levels of salicylic acid (0 and 2 mM) was conducted (2019) under control conditions in the Kermanshah Agricultural & Natural Resources Research & Education Center.
Seeds of Forest savory were obtained from the Research Institute of forests and Rangelands, Tehran, Iran. The seeds were disinfected with 0.5% sodium hypochlorite and washed and dried with distilled water. The seeds were treated with Mancozeb fungicide (2%) and sown in a mixed bed of cocopeat and peat moss (1:1) in a tray. The seeds were irrigated once a day. After seed germination, the seedlings were irrigated every three days by misting. Individual seedlings (four week olds) at 4 to 6-leaf stage (Ou et al., 2018) were moved in the main plastic pots which were filled with 4.5 kg of agricultural soil, sand, and rotted manure (1:1:1).
The plants were subjected to 17 hours of light (300 µmol/m2/s) and 7 hours of darkness, and relative humidity of 50-60%. Before the implementation of salt treatments, the plants were irrigated (once every three days) with 2500 ml of water. In order to adapt plants to salinity, the plants (except salt control plants) were irrigated twice with 20 mM NaCl before performing salt treatments.
The Merck NaCl was used for preparation of 50, 100, and 150 mM NaCl. The plants were irrigated by 250 ml of different NaCl treatments (8 weeks and twice a week). The control plants were irrigated by 250 ml distilled water. After every 4 times irrigation by salt treatments, the plants were irrigated once with distilled water to remove the salts accumulated in the pots. Salicylic acid (HOC₆H₄COOH; CAS: 69-72-7; Merck; Germany) was used to prepare a concentration of 2 mM. The plants were treated (8 weeks and once a week) with 100 ml of 2 mM SA (foliar spraying). The non-SA-treated plants were sprayed by 100 ml distilled water.
Measurement of traits
The list of trait names with their abbreviation and measurement method presented in Table 1.
Before harvesting the plants, the plant height (cm) was measured. To measure the leaf area, the 30 leaves were separated from each plant and were measured by a leaf area meter, then the average leaf area (mm2) was calculated (Andalibi et al., 2021). After harvesting the plants, the aerial part was cut from the collar and the shoot fresh weight (g) was determined by weighing. The 30 leaves of each plant were separated and immediately weighed on a scale and the average leaf fresh weight was calculated (LFW). The leaves were dried in an oven (70°C, 48 h) and the leaf dry weight (LDW) was weighed. The leaves were dried in an oven (70°C, 48 h) and the leaves dry weight was measured. Shoot dry weight (g) was measured after drying up plant aerial parts at 70°C, 48 h. The roots were gently washed, and its surface water dried with paper towels, and then the root’s fresh weight (g) was measured. The root samples dried at 75°C, 48 h and root dry weight (g) was measured.
For measurement of Fv/Fm value, 20 leaves from each pot were covered with aluminum foil and were adapted in the dark for 30 minutes. Fv/Fm was estimated using chlorophyll fluorimeter (Hansatech Pocket PEA, UK) at 695 nm. The light level (PFD photon flux level) was 400 μmol / m2 and the light radiation time was 5 seconds.
Leaf chlorophyll index (SPAD) was measured using a SPAD-502Plus, Minolta, Japan.
Proline content was measured based on (Bates et al., 1973). The OD of plant samples and proline standard was read at 520 nm by Spectrophotometer. Then the OD of each sample was put into the standard equation and was reported as μg/g FW.
Soluble protein concentration (mg/g FW) was measured based on the method of Brdford (Bradford, 1976). 1 μl of the crude leaf extract was added to 200 μl of Coomassie Brilliant Blue. After fifteen minutes, the OD of samples was read at 595 nm Spectrophotometer. The concentration of soluble protein was obtained according to the absorption of the samples and using the Bovine Serum Albumin (BSA) standard curve.
For measurement of Relative water content (RWC), 30 leaves of each plant (approximately isodiametric) were separated and immediately weighed and the average leaf fresh weight was calculated (FW). Then the leaves were immersed in double-distilled water for 16 to 18 hours (22°C). The surface of leaves was dried with filter paper and the turgor weight of leaves was weighed (TW). The leaves were dried in an oven (70°C, 48 h) and the leaf dry weight (DW) was weighed (Vaieretti et al., 2007). The RWC was calculated from equation 1 (Bian and Jiang, 2009).
Equation 1
Where:
RWC= relative water content
FW= fresh weight
DW= dry weight
TW= turgor weight
For electrical conductivity (EC), 30 leaves from each plant were separated and washed with distilled water. The leaves were immersed in 25 ml of double-distilled water (22 °C and 24 h) and the EC (μS/cm) was measured with EC meter (Blum and Ebercon, 1981).
Statistical analysis
Analysis of variance (factorial) and LSD comparison of means (Means±SD) were performed using IBM SPSS Statistics 26 software. The graphs were drawn using Excel software.
Table 1. List of trait names with their abbreviation and measurement method | |||
Name of Traits | Abbreviation | unit | Measurement method |
Plant height | PLH (cm) | cm | roller |
Leaf area | LA (cm2) | mm2 | (Andalibi et al., 2021) |
Leaf fresh weight | Leaf FW | mg | weighing |
Leaf dry weight | Leaf DW | mg | (Vaieretti et al., 2007) |
Shoot fresh weight | Shoot FW | g | weighing |
Shoot dry weight | Shoot DW | g | (Vaieretti et al., 2007) |
Root fresh weight | Root FW | g | weighing |
Root dry weight | Root DW | g | (Vaieretti et al., 2007) |
Maximum quantum yield of photosystem II | Fv/Fm | unit | chlorophyll fluorimeter |
Chlorophyll index | SPAD | unit | SPAD-502Plus |
Relative water content | RWC | % | (Bian and Jiang, 2009) |
Leaf electrical conductivity | Leaf EC | (ds/m) | (Blum and Ebercon, 1981). |
proline | proline | μg/g FW | (Bates et al., 1973) |
protein | protein | mg/g FW | (Bradford, 1976) |
Results
Morphological traits
Results of analysis of variance showed significant differences (P<0.01) between different salinity treatments for all studied traits (Table 2). Effect of SA treatments was significant for plant height, leaf area, leaf fresh weight, shoot fresh weight, root fresh weight, root dry weight (P<0.01) and Also, significant for leaf dry weight (P<0.05) (Table 2). The salinity × salicylic acid interaction effect was significant for leaf fresh weight (P<0.01). Also, this interaction effect was significant (P<0.05) for leaf dry weight, shoot fresh weight, shoot dry weight, and root dry weight (Table 2).
Plant height
The increase in salinity levels caused a decrease in plant height. The plant height was decreased by 2.05%, 8.05%, and 31.53%, respectively, in salt-treated plants with 50, 100, and 150 mM salinity treatments compared to the control plants. The highest plant height (78.33 cm) was obtained in the 2 mM SA + 100 mM NaCl, and the lowest plant height value (45.67 cm) was observed in the 150 mM NaCl (Fig. 2a). SA had increased the plant height in all the NaCl treatments (Fig. 2a).
Leaf area
The salinity levels of 100 and 150 mM significantly decreased the leaf area. The average leaf area was significantly decreased by 23.87% and 26.86% in NaCl-treated plants with the 100 and 150 mM salinity treatments, respectively, compared to the control plants (Table 3). The highest leaf area (0.91 cm2) was observed at 50 mM NaCl + 2 mM SA and the lowest leaf area (0.49 cm2) was observed at 150 mM NaCl (Fig. 2b). Salicylic acid was significantly increased in the leaf area in the SA-treated plants compared to non-SA-treated plants by 4.83, 35.62, 43.17, and 14.85% in the 0, 50, 100, and 150 mM NaCl concentrations, respectively (Fig 2b).
Leaf fresh weight and leaf dry weight
Leaf fresh weight was significantly decreased by 12.50% at 150 mM NaCl compared to the control plants (Table 3). The highest leaf fresh weight (12.97 mg) was obtained at 50 mM NaCl+ 2 mM SA and the lowest leaf fresh weight (8.67 mg) was observed at 150 mM NaCl (Fig. 2c). Salicylic acid had increased leaf fresh weight in the SA-treated plants compared to non-SA-treated plants by 28.24, 26.34, and 12.34%, respectively, in the 0, 50, and 150 mM NaCl concentrations. SA decreased the leaf fresh weight by 15.32% in the 100 mM NaCl concentration compared to the non-SA-treated plants. Leaf dry weight was decreased by 36.84 and 41.58% in the 100 and 150 mM NaCl concentrations compared to the salt control plants, respectively, compared to the salt control plants (Table 3). The highest leaf dry weight (2.39 mg) was obtained at 50 mM NaCl+ 2 mM SA and the lowest leaf dry weight (1.11 mg) was observed at 150 mM NaCl (Fig. 2d). Salicylic acid increased leaf dry weight in the SA-treated plants compared to non-SA-treated plants by 16.75, 24.45, and 7.38%, respectively in the 0, 50, and 100 mM NaCl concentrations, but it decreased leaf dry weight by 15.66% in the 150 mM NaCl concentration.
Shoot fresh weight and shoot dry weight
The shoot fresh weight was decreased by 8.82, 46.22, and 42.69% at 50, 100, and 150 mM NaCl respectively, compared to NaCl control (Table 3). The highest shoot fresh weight (24.01 g) was observed in the treatment of 0 mM NaCl+ 2 mM SA, and the lowest value (9.57 g) was observed at 100 mM NaCl (Fig. 3a). Salicylic acid increased the shoot fresh weight in the SA-treated plants by 34.87, 10.35, 42.58, and 15.69%, respectively, in the 0, 50, 100, and 150 mM NaCl concentrations, compared to the non-SA-treated plants. The shoot dry weight was decreased by 42.34, 51.34, and 69.15%, respectively, in the plants treated with the 50, 100, and 150 mM NaCl compared to the salt control plants. The highest shoot dry weight (6.73 g) was obtained at 0 mM NaCl (salt control) and the lowest it (2.03 g) was obtained in the treatment of 150 mM NaCl + 2 mM SA (Fig. 3b). Application of salicylic acid did not have significant effect on the shoot dry weight in salt control (0 mM NaCl) and high salinity treatments (100 and 150 mM NaCl), but SA increased shoot dry weight by 60.74% in the mild salt stress conditions (50 mM NaCl).
Root fresh weight and root dry weight
Root fresh weight was decreased by 62.60, 77.88, and 82.71%, respectively in the plants treated with 50, 100, and 150 mM NaCl compared to the control (Table 3). The highest root fresh weight (16.68 g) was obtained in the treatment of 0 mM NaCl+ 2 mM SA and the lowest value (2.51 g) was observed at 150 mM NaCl (Fig. 3c). Salicylic acid increased root fresh weight by 15.01, 76.34, 95.53, and 97.07%, respectively, in the 0, 50, 100, and 150 mM NaCl concentrations compared to the non-SA-treated plants. Root dry weight decreased by 62.98, 71.07, and 79.17%, respectively, in the plants treated with 50, 100, and 150 mM NaCl compared to the control plants. The highest root dry weight (3.73 g) was obtained in the treatment of 0 mM NaCl+2 mM SA and the lowest (2.07 g) was obtained at 150 mM NaCl (Fig. 3d). Salicylic acid increased the root dry weight by 9.29, 88.10, 104.11, and 88.02%, respectively in the 0, 50, 100, and 150 mM NaCl concentrations compared to the non-SA-treated plants (Fig. 3d).
Table 2. Analysis of variance of morphological traits in S. mutica under different salinity and salicylic acid (SA) treatments
Treatments | df | PLH | LA | Leaf FW | Leaf DW | Shoot FW | Shoot DW | Root FW | Root DW |
Salinity | 3 | 375.3** | 0.08 ** | 9.38 ** | 1.96 ** | 133.21** | 23.04** | 173.74** | 4.91** |
SA | 1 | 5607 ** | 0.12 ** | 8.75 ** | 0.18 * | 68.85 ** | 1.43 | 52.16 ** | 2.45** |
Salinity×SA | 3 | 73.78 | 0.02 | 6.05 ** | 0.12 * | 7.23 * | 2.37* | 1.16 | 0.13 * |
Error | 6 | 34.13 | 0.01 | 1.00 | 0.03 | 2.16 | 0.45 | 0.58 | 0.024 |
CV (%) |
| 11.35 | 15.24 | 9.60 | 10.88 | 9.70 | 17.96 | 9.64 | 9.56 |
* and **= significant at 5 and 1% probability levels, respectively. The full name of the abbreviations is presented in Table 1.
Table 3. Means± standard deviation of morphological traits in S. mutica plants under different salinity and salicylic acid (SA) treatments
Treatments | PLH (cm) | LA (cm2) | Leaf FW (mg) | Leaf DW (mg) | Shoot FW (g) | Shoot DW(g) | Root FW (g) | Root DW (g) | |
SA | 0 mM | 59.7±9.99 b | 0.58±0.11 b | 9.83±1.01 b | 1.53±0.42 b | 13.45±4.08 b | 3.98±1.87 a | 6.41±0.05 b | 1.30±0.45 b |
| 2 mM | 69.4±8.50 a | 0.72±0.16 a | 11.04±2.15 a | 1.71±0.66 a | 16.84±4.98 a | 4.47±1.01 a | 9.36±3.27 a | 1.94±0.71 a |
NaCl | 0 mM | 66.6±7.37 a | 0.67±0.12 a | 9.91±1.19 a | 1.90±0.24 a | 17.81±1.69 a | 6.73±0.59 a | 14.52±0.84a | 2.77±0.17 a |
50 mM | 65.3±5.77 a | 0.67±0.06 a | 10.43±0.52 a | 1.92±0.05 a | 16.23±2.56 a | 3.86±0.88 b | 5.42±0.89 b | 1.04±0.18 b | |
100 mM | 61.3±6.11 a | 0.51±0.08 b | 10.31±0.18 a | 1.20±0.10 b | 9.57±1.64 b | 3.26±0.50 bc | 3.21±0.33 c | 0.81±0.01bc | |
150 mM | 45.6±2.08 b | 0.49±0.03 b | 8.67±1.01 b | 1.11±0.11 b | 10.2±0.96 b | 2.06±0.59 c | 2.51±0.24 c | 0.58±0.16 c | |
Mean |
| 59.7±9.99 | 0.59±0.11 | 9.83±1.01 | 1.53±0.42 | 13.45±4.08 | 3.98±1.87 | 6.41±5.05 | 1.62±0.88 |
LSD |
| 5.60 | 0.07 | 0.74 | 0.13 | 1.61 | 0.58 | 0.57 | 0.10 |
Means of columns with the same letters are not significantly different based on LSD test. The full name of the abbreviations is presented in Table 1.
Fig. 2. Means of plant height (a), leaf area (b), leaf fresh weight (c) and leaf dry weight (d) of S. mutica plants under different treatments of NaCl×SA. Columns with the same letters are not significantly different based on LSD test (P= 0.05). The full name of the abbreviations is presented in Table 1.
Fig. 3. Means of shoot fresh weight (a), shoot dry weight (b), root fresh weight (C) and root dry weight (d) of S. mutica plants under different treatments of NaCl×SA. Columns with the same letters are not significantly different based on LSD test (P= 0.05). The full name of the abbreviations is presented in Table 1.
Physiological traits
Results of analysis of variance showed significant differences (P<0.01) between salinity treatments for all studied traits (Table 4). Significant differences between SA treatments (P<0.01) were observed for Fv/Fm, RWC, leaf electric conductivity, proline and protein content. Also, significant differences (P<0.05) were observed between SA treatments for SPAD (Table 4). The salinity by salicylic acid interaction effect were significant for SPAD, Fv/Fm, RWC, leaf electrical conductivity, proline, and soluble protein content (P<0.01) (Table 4).
Maximum quantum yield of Photosystem ІІ (Fv/Fm value)
Fv/Fm value was decreased by 4.56, 10.59, and 16.75%, respectively in the salt-treated plants with 50, 100, and 150 mM NaCl concentrations compared to control plants (Table 5). The highest Fv/Fm (0.81 units) was observed at 0 mM NaCl (control) and the lowest value (0.66 units) was observed at 150 mM NaCl (Fig. 4a). Salicylic acid increased Fv/Fm by 2.24, 4.86, 12.93, and 21.64%, respectively, at 0, 50, 100, and 150 mM NaCl concentrations compared with non-SA-treated plants (Fig 4a).
Leaf chlorophyll index (SPAD)
Increase in salinity up to 100 mM increased the amounts of chlorophyll index. SPAD was decreased by 12.7, 21.48 and 31.90%, respectively in the 50, 100, and 150 mM NaCl concentrations compared to the control plants (Table 5). The highest SPAD (36.63 units) was observed at 100 mM NaCl+2 mM SA and the lowest value (25.87 units) was observed at 150 mM NaCl (Fig. 4b). Salicylic acid enhanced leaf chlorophyll index by 19.20 and 28.6, and 23.1%, respectively, in the 50, 100, and 150 mM NaCl concentrations compared to the non-SA-treated plants (Fig. 4b).
Leaf proline content
Increase of salinity up to 100 mM, sharply increased the proline content, but 150 mM NaCl did not show any trend in the increase or decrease in proline content. The proline content was increased by 483.46, 976.92, and 976.92%, respectively in the salt-treated plants with 50, 100, and 150 mM NaCl compared with the non-NaCl-treated plants (table 5). The highest level of proline content (14.00 μg/g) was observed at 150 mM NaCl and the lowest proline content (1.00 μg/g) was observed in the 2 mM SA without salt (Fig. 4c). Salicylic acid decreased the proline content by 23.08, 71.43, 271.43, and 257.14% respectively in the 0, 50, 100, and 150 mM NaCl concentrations compared to non-SA-treated plants (Fig 4c).
Soluble protein content:
By increasing salinity up to 100 mM NaCl, the amount of leaf soluble protein increased, but it was decreased at 150 mM NaCl. Leaf soluble protein was increased by 50.00 and 80.88% in the NaCl-treated plants with 50 and 100 mM NaCl, respectively, but it decreased by 25% in the NaCl-treated plants with 150 mM NaCl compared to the non-NaCl-treated plants (Table 5). The highest soluble protein was found at 100 mM NaCl (1.23 mg/g) and the lowest value (0.51 mg/g) was observed at 150 mM NaCl (Fig. 4d). Application of salicylic acid in non-SAlt stress conditions (0 mM NaCl) increased the soluble protein by 5.88%. SA decreased the soluble protein by 26.47, 29.27, and 78.43%, respectively at 50, 100, and 150 mM NaCl concentrations compared to non-SA-treated plants (Fig 4d).
Relative Water Content (RWC)
The relative water content was decreased by 28.57, and 25.70%, respectively in the 100 and 150 mM NaCl concentrations compared to non-NaCl-treated plants (Table 5). The highest RWC (93.00%) was observed at 2 mM SA without salt and the lowest RWC (65.51%) was observed at 100 mM NaCl (Fig. 4e). Salicylic acid did not have a significant effect on the RWC in the salt control plants and plants treated with 50 mM NaCl. SA increased the RWC by 20.15 and 32.85%, respectively, in the 100 and 150 mM NaCl concentrations compared with the non-SA-treated plants (Fig 4e).
Leaf electrical conductivity (EC)
The leaf EC was increased by 56.01, 140.37, and 621.74%, respectively in the 50, 100, and 150 mM NaCl concentrations compared to the non-NaCl-treated plants (Table 5). The lowest EC (40.88 μS/cm) was observed in 2 mM SA without salt and the highest EC (324.78 μS/cm) was observed at 150 mM NaCl (Fig. 4f). Salicylic acid decreased the EC by 38.71, 14.28, 8.92, and 21.37%, respectively in the 0, 50, 100, and 150 mM NaCl concentrations compared to the non-SA-treated plants (Fig. 4f).
Table 4. Analysis of variance of physiological traits in S. mutica under different salinity and salicylic acid (SA) treatments
Treatments | df | Fv/Fm | SPAD | RWC | Leaf EC | Soluble protein | Proline |
Salinity | 3 | 0.01 ** | 271.80 ** | 553.63** | 194526.21** | 386.66** | 0.1** |
SA | 1 | 0.03 ** | 36.75 * | 463.75** | 245863.54** | 55.71** | 0.01** |
Salinity × SA | 3 | 0.01 ** | 76.72 ** | 185.37** | 48962.21** | 42.11** | 0.1** |
Error | 6 | 0.0001 | 5.19 | 8.72 | 83.29 | 2.36 | 0.002 |
CV (%) |
| 0.41 | 6.59 | 3.53 | 3.57 | 5.99 | 24.96 |
* and **= significant at 5 and 1% probability levels, respectively. The full name of the abbreviations is presented in Table 1.
Table 5. Means comparison of physiological traits in S. mutica plants under different salinity and salicylic acid (SA) treatments
Treatments | Fv/Fm (unit) | SPAD (unit) | RWC (%) | Leaf EC (μS/cm) | Proline (μg/g FW) | Protein (mg/g FW) | ||||||||
SA (mM) | 0 | 0.72±0.05 b | 29.56±7.12b | 79.2±13.18b | 137.5±11.59a | 9.75±0.18 a | 0.86±0.05 a | |||||||
2 | 0.79±0.01 a | 33.35±3.42a | 88.01±6.36a | 113.7±8.82 b | 2.00±0.08 b | 0.81±0.03 b | ||||||||
NaCl (mM) | 0 | 0.79±0.02 a | 34.10±1.59a | 91.66±0.88a | 45.4±1.98 e | 1.28±0.02 c | 0.68±0.04 c | |||||||
50 | 0.75±0.01 b | 30.27±2.92a | 91.56±0.88a | 70.2±1.97 d | 7.00±0.04 b | 1.02±0.05 b | ||||||||
100 | 0.71±0.01 c | 28.07±3.17b | 65.51±2.67b | 108.1±3.57c | 13.99±0.03a | 1.23±0.09 a | ||||||||
150 | 0.66±0.003d | 25.86±1.80b | 68.13±4.52b | 324.7±14.14a | 14.00±0.02a | 0.51±0.06 d | ||||||||
Mean |
| 0.76±0.05 | 29.56±7.12 | 83.61±11.07 | 125.3±101.38 | 4.28±0.50 | 0.84±0.24 | |||||||
LSD |
| 0.002 | 1.39 | 1.81 | 9.24 | 0.08 | 0.03 | |||||||
Means of columns with the same letters are not significantly different based on LSD test. The full name of the abbreviations is presented in Table 1.
Fig. 4. Means of Fv/Fm (a), SPAD (b), proline content (c), soluble protein content (d), RWC (e), and leaf electric conductivity (f) of S. mutica plants under different treatments of NaCl×SA. Columns with the same letters are not significantly different based on LSD test (P= 0.05). The full name of the abbreviations is presented in Table 1.
Discussion
Morphological traits were affected by salt stress. Salt stress negatively affected the leaf area and plant height earlier than other morphological traits (Su et al., 2013). In S. mutica plants an increase in salinity levels caused a decrease in plant height. In line with this finding, salinity stress has reduced the plant height in Satureja khuzestanica plants (Saadatfar and Hossein Jafari, 2022). In this study, Salicylic acid increased plant height in the SA-treated plants compared to non-SA-treated plants. In a recent study, the application of SA had increased the plant height in Lantana camara (Dehestani Ardakani et al., 2021) at different salinity levels. In present research, salinity decreased leaf area and salicylic acid increased it. Similar to our result, salinity stress had reduced the leaf area in Thymus vulgaris (Harati et al., 2015). SA had increased the leaf area in S. hortensis (Pourghadir et al., 2021).
Leaf fresh and dry weights were increased in the low salinity levels, but they decreased in the high NaCl concentrations. In line with our findings, Leaf fresh and dry weight were decreased in Pyrus betulifolia Bunge under salt stress (Yu et al., 2019). Salicylic acid had increased leaf fresh and dry weights in the SA-treated plants compared to non-SA-treated plants. In some other species such Solanum melongena L. (Mady et al., 2023) salicylic acid had increased leaf fresh and dry weights.
We found that shoot fresh and dry weight decreased in the S. mutica plants in the salt stress conditions. Also, salicylic acid significantly increased shoot fresh and dry weight. Similar to our result, shoot dry weight was decreased in different salinity levels in Amaranthus cruentus (Menezes et al., 2017). Salicylic acid had improved the shoot dry weight in Lantana camara at high salinity levels compared to the control (Dehestani Ardakani et al., 2021). These results were consistent with our observations.
The root fresh and dry weights were decreased in the plants treated with NaCl. Similarly, a decrease in root dry weight has been observed in Amaranthus cruentus plants treated with 50, 75, and 100 mM NaCl (Menezes et al., 2017). Salicylic acid increased root fresh and dry weight in the SA-treated plants under salt stress conditions. Similar to this, Salicylic acid has increased the root fresh and dry weight in Thymus vulgaris plants under salinity stress conditions (Harati et al., 2015).
Chlorophyll index is a proper factor to evaluate the plant's photosynthetic potential (Yang et al., 2023). In our research, chlorophyll index (SPAD) was significantly decreased in the high NaCl concentrations. (Khattab, 2007)(Khattab 2007)In Satureja khuzestanica plants the chlorophyll content has significantly been reduced by increasing the salt concentration (Saadatfar and Hossein Jafari, 2023). Salicylic acid increased leaf chlorophyll index in the SA-treated S. mutica plants at all NaCl concentrations compared to the non-SA-treated plants. In some other plants such as St. John's wort, salicylic acid had increased the photosynthetic index under salt stress conditions. (Kwon et al., 2023)
Fv/Fm value decreased in the NaCl-treated plants compared to control. High levels of salinity had decreased Fv/Fm in Nitere Bush (Nitraria schoberi L.) Plants (Ranjbar-Fordoei and Dehghani-Bidgoli, 2016). Salicylic acid increased Fv/Fm in the SA-treated plants compared with non-SA-treated plants in all NaCl concentrations. In line with our finding, salicylic acid had increased the Fv/Fm in Hypericum perforatum (Kwon et al., 2023) under salinity stress conditions.
The relative water content (RWC) was decreased in the salt-treated plants and SA increased its value in the SA-treated plants compared to the non-SA-treated plants. In Trigonella foenum-graecum L. (Arvin and Firuzeh, 2021) salinity had decreased RWC. Application of salicylic acid had increased the RWC in Lantana camara (Dehestani Ardakani et al., 2021) under salinity conditions.
Salinity stress critically increased leaf electrical conductivity and salicylic acid decreased it under salt stress conditions. In line with this finding, salt stress had increased the relative electrical conductivity (REC) in Dianthus superbus plants, and the application of salicylic acid effectively reduced it (Ma et al., 2017).
In salinity stress, plants adjust their osmotic potential by accumulating osmolytes to escape from dehydration. In the present study, the proline content was increased significantly by salinity levels. Similarly, proline content had significantly increased by rising salt levels in Satureja khuzestanica (Saadatfar and Hossein Jafari, 2023).
Salicylic acid significantly reduced proline content in the S. mutica under severe salt stress conditions. Similar to our result, SA application had reduced proline content in Nigella sativa L. (Zarei et al., 2019) under salt stress conditions.
By increasing salinity up to 100 mM NaCl, the amounts of leaf soluble protein were increased, but it value was decreased at 150 mM NaCl concentrations. Protein content in Thymus vulgaris was increased in mild salt treatments and decreased in severe salt stress conditions (Harati et al., 2015). In our research, application of salicylic acid had increased the soluble protein content in control and low and moderate salt stress conditions, but it decreased the soluble protein in high salt stress conditions. Salicylic acid had increased leaf soluble protein under low and moderate salt stress conditions in Thymus vulgaris (Harati et al., 2015). These results confirm the validity of our findings.
Conclusion
Salinity stress decreased the relative water content, and increased the electrical conductivity of the cell, subsequently disturbed the physiological, photosynthetic and biochemical functions of the cell, and as a result, reduced the growth and yield of the Forest savory plants. Application of 2 mM salicylic acid modulated the effects of salinity stress by increasing the relative water content and improved the physiological, photosynthetic and biochemical functions of the cell, and subsequently increased plant growth and yield. The threshold of salinity tolerance of forest savory is less than 100 mM, and in soils more salty than this value, its yield significantly decreases. Foliar spraying with 2 mM salicylic acid (four times) in the early stages of vegetative growth, increases the plant's tolerance to salinity and significantly increases its yield in low salinity and mild salinity soils.
Acknowledgment
We are grateful to the colleagues of Kermanshah Agricultural and Natural Resources Research and Education Center, Iran who sincerely cooperated in the implementation of this research.
References
1. Andalibi, L., Ghorbani, A., Moameri, M., Hazbavi, Z., Nothdurft, A., Jafari, R. & Dadjou, F. 2021. Leaf area index variations in ecoregions of Ardabil province, Iran. Remote Sensing, 13, 2879.
2. Arif, Y., Singh, P., Siddiqui, H., Bajguz, A. & Hayat, S. 2020. Salinity induced physiological and biochemical changes in plants: An omic approach towards salt stress tolerance. Plant Physiology and Biochemistry, 156, 64-77.
3. Arvin, P. & Firuzeh, R. 2021. Effects of salinity stress on physiological and biochemical traits of some fenugreek (Trigonella foenum-graecum L.) populations. Iranian Journal of Medicinal and Aromatic Plants, 37, 822-837 (In farsi).
4. Athar, H. U. R., Zulfiqar, F., Moosa, A., Ashraf, M., Zafar, Z. U., Zhang, L., Ahmed, N., Kalaji, H. M., Nafees, M., Hossain, M. A., Islam, M. S., EL sabagh, A. & Siddique, K. H. M. 2022. Salt stress proteins in plants: An overview. Frontiers in Plant Science, 13, doi:10.3389/fpls.2022.999058.
5. Bates, L. S., Waldren, R. P. & Teare, I. D. 1973. Rapid determination of free proline for water-stress studies. Plant and Soil, 39, 205-207.
6. Behzadi Rad, P., Roozban, M. R., Karimi, S., Ghahremani, R. & Vahdati, K. 2021. Osmolyte accumulation and sodium compartmentation has a key role in salinity tolerance of pistachios rootstocks. Agriculture, 11, 708.
7. Bertamini, M., Grando, M., Zocca, P., Pedrotti, M., Lorenzi, S. & Cappellin, L. 2019. Linking monoterpenes and abiotic stress resistance in grapevines. BIO Web of Conferences, 13, 01003, https://doi.org/10.1051/bioconf/20191301003.
8. Betzen, B., Smart, C., Maricle, K. & Maricle, B. 2019. Effects of increasing salinity on photosynthesis and plant water potential in kansas salt marsh species. Transactions of the Kansas. Academy of Science, 122, 49.
9. Bian, S. & Jiang, Y. 2009. Reactive oxygen species, antioxidant enzyme activities and gene expression patterns in leaves and roots of Kentucky bluegrass in response to drought stress and recovery. Scientia Horticulturae, 120, 264-270.
10. 10 Blum, A. & Ebercon, A. 1981. Cell membrane stability as a measure of drought and heat tolerance in Wheat. Crop Science, 21, doi: 0011183X002100010013x.
11. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254.
12. Dehestani Ardakani, M., Ghatei, P., Gholamnezhad, J., Momenpour, A. & Fakharipour Charkhabi, Z. 2021. Improving growth and physiological characteristics in salt stressed lantana (Lantana camara Linn.) by application of exogenous salicylic acid. Journal of Agricultural Science and Sustainable Production, 31, 95-115.
13. Harati, E., Kashefi, B., Matinzadeh & M 2015. Investigation Reducing Detrimental Effects of Salt Stress on Morphological and Physiological Traits of (Thymus vulgaris) by Application of Salicylic Acid. Iranian Journal of plant physiology, 5, 1383-1391 (In Persian).
14. Imani, M., Ghasemi, H., Sobhani Rad, S. & Behgar, M. 2021. Different levels of clove and Marze essential oils on fermentation parameters in gas production technique and their antimicrobial effects on Peptostreptococcus anaerobius bacteria isolated from the rumen. Iranian Animal Science Research, 13, 335-349 (In Persian).
15. Karimi, A., Gholamalipour Alamdar, E., Avarseji, Z. & Nakhzari Moghaddam, A. 2021. The effect of planting density and number of hand weeding times on weeds control and relationship between morphological characteristics with content and yield of essential oil of Satureja hortensis L. . Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, 69, 417-426.
16. Kaya, C., Ugurlar, F., Ashraf, M. & Ahmad, P. 2023. Salicylic acid interacts with other plant growth regulators and signal molecules in response to stressful environments in plants. Plant Physiology and Biochemistry, 196, 431-443.
17. Krause, S. T., Liao, P., Crocoll, C., Boachon, B., Förster, C., Leidecker, F., Wiese, N., Zhao, D., Wood, J. C., Buell, C. R., Gershenzon, J., Dudareva, N. & Degenhardt, J. 2021. The biosynthesis of thymol, carvacrol, and thymohydroquinone in Lamiaceae proceeds via cytochrome P450s and a short-chain dehydrogenase. Proceedings of the National Academy of Sciences, United States of America, 118, e2110092118, https://doi.org/10.1073/pnas.2110092118
18. Kwon, E.-H., Adhikari, A., Imran, M., Lee, D.-S., Lee, C.-Y., Kang, S.-M. & Lee, I.-J. 2023. Exogenous SA applications alleviate salinity stress via physiological and biochemical changes in st john’s Wort Plants. Plants, 12, 310.
19. Ma, X., Zheng, J., Zhang, X., Hu, Q. & Qian, R. 2017. Salicylic acid alleviates the adverse effects of salt stress on Dianthus superbus (Caryophyllaceae) by activating photosynthesis, protecting morphological structure, and enhancing the antioxidant system. Frontiers in Plant Science, 8. https://doi.org/10.3389/fpls.2017.00600
20. Mady, E., Abd EL-Wahed, A. H. M., Awad, A. H., Asar, T. O., AL-Farga, A., Abd EL-Raouf, H. S., Randhir, R., Alnuzaili, E. S., EL-Taher, A. M., Randhir, T. O. & Hamada, F. A. 2023. Evaluation of salicylic acid effects on growth, biochemical, yield, and anatomical characteristics of Eggplant (Solanum melongena L.) plants under salt stress conditions. Agronomy, 13, 2213.
21. Mahajan, M. & Pal, P. K. 2023. Chapter Five - Drought and salinity stress in medicinal and aromatic plants: Physiological response, adaptive mechanism, management/amelioration strategies, and an opportunity for production of bioactive compounds. In: Sparks, D. L. (ed.) Advances in Agronomy, Academic Press. https://10.1016/bs.agron.2023.06.005.
22. Menezes, R. V., Neto, A. D. A., Ribeiro, M. O. & Cova, A. M. W. 2017. Growth and contents of organic and inorganic solutes in amaranth under salt stress. Agropecuária Tropical Goiania, 47, 22-30.
23. Mushtaq, Z., Faizan, S., Gulzar, B., Mushtaq, H., Bushra, S., Hussain, A. & Hakeem, K. 2022. Changes in growth, photosynthetic pigments, cell viability, lipid peroxidation and antioxidant defense system in two varieties of Chickpea (Cicer arietinum L.) subjected to salinity stress. Phyton, 91, 149. https://10.32604/phyton.2021.016231
24. Ou, J., Thompson, C. R., Stahlman, P. W., Bloedow, N. & Jugulam, M. 2018. Reduced translocation of glyphosate and dicamba in combination contributes to poor control of Kochia scoparia: Evidence of Herbicide Antagonism. Scientific Reportes, 8, 5330.
25. Nieto, G. 2020. A review on applications and uses of thymus in the food industry. Plants, 9, 961.
26. Pourghadir, M., Mirjalili, S. A., Mohammadi Torkashvand, A. & Moradi, P. 2021. Growth, essential oil yield and components of summer savory (Satureja hortensis L.) influenced by salicylic acid and proline. Iranian Journal of Plant Physiology, 11, 3863-3872.
27. Ranjbar-Fordoei, A. & Dehghani-Bidgoli, R. 2016. Impact of salinity stress on photochemical efficiency of photosystem II, chlorophyll content and nutrient elements of Nitere Bush (Nitraria schoberi L.) plants. Journal of Rangeland Science, 6, 1-9.
28. Saadatfar, A. & Hossein Jafari, S. 2022. The effect of methyl jasmonate on morpho-physiological and biochemical parameters and mineral contents in Satureja khuzistanica Jamzad under salinity stress. Journal of Medicinal Plants, 21, 87-99.
29. Saadatfar, A. & Hossein Jafari, S. 2023. Application of 24-epibrassinolide as an environmentally friendly strategy alleviates negative effects of salinity stress in Satureja khuzistanica Jamzad. Journal of Rangeland Science, Articles in Press, Available Online from 31 July 2023.
30. Stahl-Biskup, E. 2002. Essential oil chemistry of the genus Thymus–a global view, 1st Edition, CRC press, Florida, United States, 50 Pages.
31. Su, J., Wu, S., Xu, Z., Qiu, S., Luo, T., Yang, Y., Chen, Q., Xia, Y., Zou, S., Huang, B.-L. & Huang, B. 2013. Comparison of salt tolerance in Brassicas and some related species. American Journal of Plant Sciences, 4(10), 1911-1917.
32. Vaieretti, M. V., Díaz, S., Vile, D. & Garnier, E. 2007. Two measurement methods of leaf dry matter content produce similar results in a broad range of species. Annals of Botany, 99, 955-8.
33. Wang, C., Gu, Q., Zhao, L., Li, C., Ren, J. & Zhang, J. 2022. Photochemical efficiency of photosystem II in inverted leaves of Soybean [Glycine max (L.) Merr.] affected by elevated temperature and high light. Frontiers in Plant Science, 12. https://doi.org/10.3389%2Ffpls.2021.772644
34. Wang, L., Pan, D., Li, J., Tan, F., Hoffmann-Benning, S., Liang, W. & Chen, W. 2015. Proteomic analysis of changes in the Kandelia candel chloroplast proteins reveals pathways associated with salt tolerance. Plant Science, 231, 159-72.
35. Yang, Y., Nan, R., Mi, T., Song, Y., Shi, F., Liu, X., Wang, Y., Sun, F., Xi, Y. & Zhang, C. 2023. Rapid and nondestructive evaluation of Wheat chlorophyll under drought stress using hyperspectral imaging. International Journal of Molecular Science, 24 (6), 5825. https://doi.org/10.3390/ijms24065825
36. Yu, X., Shi, P., Hui, C., Miao, L., Liu, C., Zhang, Q. & Feng, C. 2019. Effects of salt stress on the leaf shape and scaling of Pyrus betulifolia Bunge. Symmetry, 11, 991.
37. Zarei, B., Fazeli, A. & Tahmasebi, Z. 2019. Salicylic acid in reducing effect of salinity on some growth parameters of Black cumin (Nigella sativa). Plant Process and Function, 8, 287-298.
38. Zuo, S., Zuo, Y., Gu, W., Wei, S. & Li, J. 2022. Exogenous proline optimizes osmotic adjustment substances and active oxygen metabolism of Maize embryo under low-temperature stress and metabolomic analysis. Processes, 10, 7, 1388. https://doi.org/10.3390/pr10071388
اثر تنش شوری و اسید سالیسیلیک بر خصوصیات مورفولوژیکی، فیزیولوژیکی و رشد گیاه مرزه سفید (Satureja mutica Fisch. & C. A. Mey)
برزو یوسفیالف * و رویا کرمیان ب
الف دانشجوی دکتری PhD، گروه زیست شناسی، دانشکده علوم، دانشگاه بوعلی سینا، همدان، ایران.
*(نویسنده مسئول)، ایمیل: borzooyoosefi@yahoo.com
ب استاد ، گروه زیست شناسی، دانشکده علوم، دانشگاه بوعلی سینا، همدان، ایران.
چکیده
مرزه سفید گیاهی خودرو، مرتعی و چندساله است که از شمال غرب تا شمال شرق ایران رویش دارد و در صنایع بهداشتی و محصولات غذایی کاربرد دارد. به منظور بررسی اثر تعدیل کنندگی سالیسیلیک اسید بر صفات مرفوفیزیولوژیکی گیاه مرزه جنگلی در شرایط تنش شوری آزمایشی گلدانی به صورت فاکتوریل در قالب طرح کاملاً تصادفی (CRD) و سه تکرار در مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی کرمانشاه در سال 1398 انجام شد. فاکتور A شامل 4 غلظت شوری (0، 50، 100 و 150 میلیمولار نمک طعام) و فاکتور B شامل 2 غلظت اسید سالیسیلیک (0 و 2 میلیمولار) بود. نتایج نشان داد که اسید سالیسیلیک به طور متوسط صفات ارتفاع بوته (54/17%)، سطح برگ (62/24%)، وزن تر برگ (12/41%)، وزن خشک برگ (23/8%)، وزن تر اندام هوایی (87/25%)، وزن خشک اندام هوایی (75/13%)، وزن تر ریشه (99/70%)، وزن خشک ریشه (38/72%) را نسبت گیاهان تیمار نشده افزایش داد. برای صفات فیزیولوژیکی تیمار اسید سالیسیلیک محتوای آب نسبی (85/32%) حداکثر عملکرد کوانتومی فتوسیستم П (42/10%) و شاخص کلروفیل را (36/11%) را نسبت گیاهان تیمار نشده با SA افزایش داد. در مقابل، هدایت الکتریکی برگ (37/2%) و محتوای پرولین (07/12%) را کاهش داد. آستانه تحمل به شوری مرزه جنگلی کمتر از 100 میلیمولار است و در خاکهای شورتر از این مقدار، عملکرد آن به میزان قابل توجهی کاهش مییابد. محلولپاشی با اسید سالیسیلیک 2 میلیمولار در مراحل اولیه رشد رویشی در گیاه مرزه جنگلی تحت تنش شوری تحمل گیاه را به شوری ارتقاء داد و عملکرد آن را در شرایط شوری کم و ملایم به میزان قابل توجهی افزایش داد.
کلیدواژگان: پرولین، تنش شوری، فتوسنتز، فلورسانس کلروفیل، شاخص کلروفیل، مرزه
