Physiological, morphological and phytochemical responses of ajowan (Trachyspermum ammi L.) populations to salt stress

Twenty-eight populations of ajowan (Trachyspermum ammi L.) were evaluated for morphological traits and oil yield in two consecutive years. Then, selected ajowan populations that revealed medium and high oil yield and higher seed weight in two years were selected for further studies. These were assessed for physiological traits, total phenolic and avonoid contents and antioxidant capacity under four salt treatments control, 60, 90, and 120 mM NaCl. The essential oil composition was analyzed by gas chromatography-mass spectrometry (GC-MS) Thymol (32.7-54.29%), γ -terpinene (21.71–32.81%), and p-cymene (18.74–26.16%) were major components. The highest and lowest thymol were recorded for Qazvin (control) and Qazvin (Low salt concentration), respectively. Salt stress caused an increase in essential oil content of Esfahfo and Qazvin populations. The highest phenolic and avonoid contents were found in Arak population grown in 60 mM NaCl (183.83 mg TAE g − 1 DW) and Yazd population grown in 90 mM NaCl (5.94 mg QE g − 1 DW). Moreover, Yazd population exhibited the strongest antioxidant activity based on DPPH (IC 50 = 1566µg/mL) under 60 mM NaCl and the highest reducing power (0.69 nm) under 120 mM NaCl. Overall, the results revealed that low and moderate salt stress improves the phytochemicals of ajowan, being especially useful for pharmaceutical and food applications. Kazerun, (48.07%), (33.73%), (17.41%)


Introduction
A growing interest is nowadays being shown to replacing synthetic aromatic compounds with counterpart natural extracts. Distributed in semi-arid and arid parts of the world 1 , ajowan, or sprague, (Trachyspermum ammi L.) is a medicinal and industrial plant of the Apiaceae family with white owers and small brownish fruits. A good source of secondary metabolites, the seeds of ajowan have been used in food and pharmaceutical applications 2 . A variety of health properties was described for the ajowan seeds, which include antimicrobial, antioxidant, nematicidal, anti-in ammatory, carminative, and sedative effects 2 . Thymol, is considered to be a valuable phenolic monoterpene that is widely used in food products for its high antioxidant and antimicrobial capacity 3 .Interestingly, ajowan seeds have also been found to be a good source of thymol. The seeds are suitably used for improving certain food products, especially due to their resistance to processing and their health-promoting compounds.
Phenolic compounds are among the secondary metabolites of medicinal plants that play crucial role in scavenging free radicals 4 . That means, the expression of these compounds can be regulated or induced by abiotic stress like salinity which is a major problem in the arid and semi-arid regions 5,6 . It could, therefore, be a major objective of the food industry to develop methods that would elevate the amounts of desirable metabolites in the medicinal plants.
Furthermore, physiological processes can also highly be affected by salt stress. Ajowan seed is of prime economic importance, and hence, its phenolic compounds as non-enzymatic antioxidants and enzymatic antioxidants play dual role on one hand they serve as a critical health-promoting constituents in human nutrition on the other.
A number of studies have been so far conducted on variations in the essential oil content of ajowan populations 1,2 , while a few concentrated on the polyphenols and antioxidant activity of the oils 2 . The effects of salt stress have been explored in some medicinal plants including Myrthus communis 6 , Mentha canadensis 7 , Thymus vulgaris 8 , and Salvia mirzayanii 9 , Nonetheless, There is scant information available in ajowan on the effects of salt stress on physiological and biochemical attributes.
The present study was, therefore, conducted: (1) to assess the morphological, oil and yield related traits in 28 ajowan populations in two consecutive years; (2) to evaluate the effects of salt stress on the essential oil content and compounds of four selected ajowan populations; (3) to assess the phenolic and avonoid, antioxidant activities and physiological traits of the studied populations under salt stress.

Results And Discussion
Two year analyses in all populations. The results of two-year analysis of variance revealed signi cant differences among the studied genotypes for all traits.
Also, the effect of year was signi cant for all. The interaction of G*Y (Genotype×Year) was also signi cant for all the studied ones (Table 1).
During the rst agronomic year, plant height varied from 63.66 to 100.5 cm ( Table 2). The tallest population was Khormo, while the shortest one was Farsfars population. Number of owering branches ranged from 6.16 (Ardebil) to 20.83 (Khormo). Arak and Farsfars population recorded the lowest in orescence diameter, while IPK2, and Khorsar population possessed the highest in orescence diameter. Number of umbel varied considerably from 29.83 to 273.39 (Table   2). IPK3 and Farsfars possessed the highest and the lowest number of umbellule per in orescence, respectively. Number of owers per umbel ranged from 114 (Farsfars) to 282.67 (Yazshah). The highest and the lowest number of seeds per umbel were recorded for Yazshah (565.33) and Farsfars (228), respectively. The lowest (278.6 cm 2 ) and the highest (1548.7 cm 2 ) crown cover diameter belonged to Khorbi and Arakkho population. The two genotypes of Yazsad and Khormo exhibited the highest values for one thousand seed weight and seed yield per plant (0.96 g and 72.45, respectively), while Ardebil (0.67 g) and Tehran (9.85 g) recorded the lowest values. The highest and the lowest essential oil yield were recorded for Yazd (5.51) and Farsfars (1.20), respectively.
In the second agronomic year, plant height varied from 87.83 cm to 139.67 cm. The highest number of owering branch was measured in the population Ardebil, while the lowest (10.5) was measured for Hamdan (Table 2). In orescence diameter varied from 3.3 cm to 5.5 cm. Number of umbel varied from IPK1 (76) to Ardebil (550. 3). Esfahfo recorded the highest number of umbellule per in orescence, while Khorbi recorded the lowest one. Number of owers per umbel ranged from 164.6 (IPK1) to 510.1 (Qazvin). Qazvin and IPK1 exhibited the highest and lowest number of seeds per umbel, respectively. Crown cover diameter ranged from 489.1 cm 2 (Khorbi) to 1896.9 cm 2 (Khorsar). Yazist had the lowest one thousand seed weight (0.61 g), while Arakkho recorded the highest (0.96 g). The highest and lowest seed yield per plant were recorded for Esfahfo (315.02 g) and IPK1 (19.30 g). Finally, the highest essential oil yield was observed in Yazd (5.58) while the lowest belonged to Yazshah. Similar ranges were also reported for Indian ajowan populations 10 . High variation in two studied years can be resulted from environmental uctuations as previously reported in other Apiaceae plants including ajowan 11 , fennel 12 and cumin 13 .
Hierarchical cluste analysis. The clustering patterns of the 28 ajowan (Trachyspermum ammi L.) populations based on their morphological data and oil yield obtained from the Ward method for two years are presented in Fig. 1 and Fig. 2. Using the analytical results of the two-year data, the genotypes were grouped into two clusters. In the rst year, group 1, included Khorsar, Arakkho, Yazsad, Yazd, Esfahfo, Farsmar and Ardebil populations with the highest essential oil yield, while group 2 classi ed 16 populations with moderate to high range of one thousand seed weight. Five populations including Yazshah, Esfahgh, IPK3, Hamadan and Khormo were placed in group 3 was characterized mostly by high plant height.
In the second year, group 1. Group 2 consisted of 11 populations. Group 3, included Ardebil, Hamadan, Esfahgh, Araksha and Khorsa populations with high plant height. Group 4 consisted of four populations with mostly by moderate one thousand seed weight, while group 5 was characterized by high essential oil yield (Fig. 2).
As the results of two years analysis revealed high variation in respect to most of the traits. So, the major criteria for selection of populations for next treatments were the economical and industrial ones. So, for this purpose, essential oil yield and one thousand seed weight were used for selections.
Accordingly, two populations that showed medium (Esfahfo and Qazvin) and two (Arak and Yazd) that revealed high amount of the mentioned traits were chosen for further experiments ( Table 2).
The results of salt treatments on selected populations Essential oil content. Based on the results obtained, the oil contents of the T. ammi populations were found to be considerably in uenced by the salt stress treatments, While essential oil yield varied from 2.16 to 4.77% under the control condition, the highest and lowest yields were recorded for Arak and Qazvin, respectively, both of which exhibited strongly reduced levels under the examined NaCl concentrations (  (Table 3).
Previous studies reported different ranges of essential oil yield for different ajowan populations collected from different countries. Chauhan et al. 14 reported a range of 2 to 4% for the essential oil yield extracted from ajowan seeds. These results are con rmed by Bairwa et al. 15 who also reported a range of 2-4.4% for the essential oil yield extracted by hydro-distillation from some Iranian ajowan populations. This is while higher ranges of 2.5-6.1% have also been reported for EO yields of Iranian ajowan populations 16 . In the present experiment, salinity stress was observed to decrease the essential oil yield in Yazd and Arak populations. This might have been due to the additional energy demand by plant tissues as a result of less available carbon concentration during the growth stage that results in reduced oil accumulation 7 . Furthermore, the increased production of volatile compounds in Esfahfo and Qazvin under elevated salt stress could be attributed to the elevations of oil gland density in these populations 17 .
The effect of salt stress on thymol content has also been reported in the genus Thymus 8 . This is while the populations examined in the present study showed different trends of thymol accumulation in their seeds. The differences observed between thyme and ajowan plants with respect to their thymol content might be attributed to their harvested organs. While it is the seeds of the ajowan species that are harvested for their high thymol content, the edible leaves of Thymus are harvested for thymol extraction.
Thymol is an aromatic and oxygenated monoterpene. Furthermore, monoterpene accumulation can be highly affected by not only phenological stages but by harvesting time as well 15 . In the case of ajowan, seeds are harvested at full maturity and monoterpenes mostly begin to increase from the full owering stage to seed maturity 2 . Moreover, salt stress reportedly affects the biosynthesis of isoprenoids as a result of its in uence on isoprene subunits.
The different trends observed in thymol accumulation in the populations examined in the present study make it di cult to draw de nitive conclusions about its quantity in different populations. However, comparison of the control and severe stress treatments revealed that Arak, Esfahfo, and Qazvin showed decreases in their thymol contents in the severe stress treatment. A number of explanations have been put forth for the decrease in thymol content. One explanation claims the dissipation energy mechanism involved in isoprenoid changes under stress conditions to be responsible as the changes are attributed to the subunits available for the biosynthesis of isoprenoids or the related compounds 4 . Furthermore, it has been argued that plants subjected to severe stress (SS) prefer to use the available carbon sources for the production of carbohydrates that are necessary for grain lling 21 . Finally, the radical scavenging mechanism has also been suggested for changes in metabolites during stress conditions 4,16 . Whatever the explanation, certain compounds with high antioxidant activities might be involved in order to cope with free radicals.
Number of seeds per plant. According to results obtained, the number of seeds per plant of the T. ammi populations were found to be considerably in uenced by the salt stress treatments. Salt stress caused a signi cant reduction in seed yield per plant of T. ammi (  (Table 3). NaCl in level modarate stress and severe stress in the growth medium caused a marked reduction in number of seeds per plant in T. ammi. Such an adverse effect of salinity on growth and seed yield has earlier been observed in a number crops, e.g. alfalfa 22,23 , carrot 24 , and cumin 25 .
Total phenolic and avonoid contents. The ajowan populations studied also showed different trends with respect to their accumulation of phenolic compounds. All the samples exhibited high TPC values ranging from 61.76 in Yazd (C) to 183.83 mg TAE g -1 DW in Arak (LS) followed by Qazvin (LS) (157.32 mg TAE g -1 DW). The accumulation of phenolic compounds in each plant is the result of such varied parameters as phenological stage, extraction process, agricultural application, and storage conditions 26 . In plants exposed to abiotic stresses, the rate of cellular oxidative damage can be controlled by the plant's capacity to produce antioxidants 27 . However, accumulation of phenolic compounds might be different in different plants as a result of salinity stress. For instance, phenolic compounds were shown to decrease in broccoli 28 . In response to salt stress, whereas NaCl treatment elevated TPC levels in maize 29 and red pepper 30 .
Similarly, the ajowan populations studied exhibited substantial differences with regard to their avonoid content. While Yazd (MS) recorded the highest TFC content (5.94 mg QE g -1 DW), Yazd (C) exhibited the lowest (3.48 mg QE g -1 DW). Moreover, a signi cant increase was observed in TFC under moderate salinity stress (MS) but higher salt concentrations was observed to cause diminishing levels of TFC (Table 3).
Plants are reported to employ different mechanisms for distributing avonoids among their subcellular sections. Metabolically, plant polyphenols, such as avonoids and phenolics, are biosynthesized through several pathways 4 . The underlying mechanism involved in avonoid functions is based on the chelating or chipping process. Some reports evidenced the enhancement of phenolic in various plant structures and organ systems under salinity stress condition 27 . It is thought that moderate salinity stress induces the normal saline tolerance pathway via increasing avonoid contents 30 . Hence, the variations observed in the studied ajowan populations as well as the different salt stress conditions might have led to the increase in the polymorphism in avonoids and their accumulation.
Antioxidant activity DPPH assay. Comparisons were made to detect variations in the scavenging of DPPH free radicals in the studied ajowan extracts ( Figure. 3). The IC 50 values were found to vary from 1566.985 µg/mL to 5889.99 µg/mL. More speci cally, the extract from the Yazd population subjected to MS and SS showed the strongest antioxidant activities (1566.985 and 1657.46 µg/mL, respectively), while those from Qazvin (C) and Esfahfo (C) demonstrated the weakest activities (5889.99 and 5671.98 µg/mL, respectively). The variation in IC 50 observed among different species might be interpreted with recourse to the diversity in their polyphenolic components 14 . Probably be suggested that plants activate metabolite biosynthesis as part of a complex antioxidant defense mechanism when they are exposed to salt stress and that the production of phenolic compounds might be part of an alternative strategy adopted by plants to respond to stressful conditions. The antioxidant capacity of Thymus species has been well researched. The most relevant chemotypes of Thymus species have been reported to be rich in phenolic monoterpenes such as thymol and carvacrol 31 . In most such studies, phenolics, due to their chemical structures that allow them to donate hydrogen to free radicals, were introduced as the major factor contributing to the antioxidant activity of the species 32 . Moreover, Tohidi et al. 33 reported that based on the observations, the highest antioxidant capacity of the recorded might be due to its high amounts of phenolic components.
Studying the effect of salt stress on the antioxidant activity of Apiaceae plants, Pandey et al. 10 (Figure 6). Based on the structural similarity of thymol and carvacrol, it may be suggested that the rate of their conversion to each other may be affected by such environmental factors as salt stress 21 .
PCA was also carried out to group the investigated populations in terms of their major oil components and the other studied metabolites. The PCA result revealed that the rst and second components explained 69.32% of all the variation observed (Table 5) while the rst component showed 44.40% of the total variation. Finally, PC2 explained 24.91% of the total variance. The rst PC (PC1) had a positive correlation with p-cymene (0.480) and y-terpinene (0.533), but a negative one with Thymol (-0.580). It may be noted that thymol is an isomer of carvacrol while p-cymene is considered as the precursor to both compounds 36 . Finally, PC2 showed a high positive contribution by EO content (0.405) and thymol (0.220) but negative correlation with phenol (-0.690). Comparison of cluster and PCA results showed the similar trends in most cases such as Yazd (SS), Esfahfo (C), Arak (C) and Arak (SS) possessed high amount of essential oil yield. As well as, Qazvin population in control conditions was rich in thymol. The seven population of Qazvin (SS), Yazd (C), Esfahfo (LS), Qazvin (LS) formed a single group characterized by higher of p-cymene and γ-terpinene. The four population of Qazvin (SS), Arak (SS), Arak (MS), Yazd (LS), Esfahfo (MS), Esfahfo (LS), and Arak (LS) formed a single group characterized by higher of TPC. Overall, the studied ajowan populations exposed to different salt level concentration and control condition were successfully distinguished based on their phytochemical traits and main EO components.
Correlations among the components. Correlation analysis demonstrated the relationships between all the measured traits and the main essential oil components under different conditions. According to Table 6, at Control condition, negative correlations were recorded between thymol and γ-terpinene correlation was shown between γ-terpinene and p-cymene (0.944 * ), and EO content and p-cymene ( Table 7). The γ-terpinene is the major precursor to the biosynthesis of thymol, p-cymene is considered as a by-product in this pathway 33 . Previous reports have shown different trends in the accumulation of these components in thyme leaves 33 . As the seeds of ajowan were used in the present study to analyze for essential oil analysis, the discrepancies observed between the results obtained for thyme and ajowan might be explained with recourse to the organs in which the oils accumulated. This is con rmed by the results reported elsewhere that highlighted changes in monoterpene frequencies based on phenological differences and harvested organs 37 (Table 3). Hydrogen peroxide (H 2 O 2 ) is an active signal molecule and its accumulation leads to a wide range of plant responses to environmental stresses, as these reactions are interdependent 41 . Increasing the level of environmental stresses increase the production of reactive oxygen species (ROS) such as hydrogen peroxide that lead to increase in damage to plant cells 42 . In the present study, the selected ajowan populations revealed high physiological variation against salt stress. Previous studies also highlighted this fact that different species and populations can reveal different reactions against stress and release various types of antioxidants that neutralize the effect of signal molecules and increase plant tolerance to stress 43 . Salinity tolerant cultivars have less hydrogen peroxide than sensitive cultivar. Therefore, leaf hydrogen peroxide content under stress conditions can be used as a suitable indicator for selection in salinity tolerance. This kind of variation was also observed in Apiaceae plants including Carum carvi L. 44 and Foeniculum vulgare Mill 45 .
Antioxidant enzymes activity. The results of statistical analysis showed that there is a signi cant difference among the populations, different salinity levels and the interaction of the populations in salinity on the activity of guaiacol peroxidase and ascorbate peroxidase (  (Table 3). Also, the highest amount of ascorbate peroxidase enzyme was related to Arak populations in control and Arak conditions at a stress level of 6 dS / m with 0.025 FW U mg -1 , respectively. The lowest was related to Yazd populations at a stress level of 6 dS / m and Qazvin the stress level of 9 dS / m ( Table 3).
According to ANOVA, the main effect of populations, salinity and the interaction of populations on salinity were signi cant for chlorophyll a and chlorophyll b ( Table 8). Interaction of salinity effects in populations for chlorophyll a revealed that the highest and lowest rates of this trait were related to Esfahfo populations at a stress level of 9 dS / m with 0.36 mg /g and Yazd populations at a stress level of 6 dS / m with 0.05 mg / g, respectively (Table 3). Also, the interaction of salinity effects in the populations for chlorophyll b showed that the highest and lowest values of this trait were related to Isfahfo populations at a stress level of 9 dS / m with 0.09 mg/g and Yazd in 9 dS / m conditions (0.023 mg / g), respectively (Table 3). Due to the direct role of chlorophyll a in photosynthesis and dry matter production, this trait can also be effective increasing this difference. Most of previous reports indicated that the chlorophyll content decreases under salinity stress and the old and necrotic leaves begin to fall as the salinity period continues. Decreases in chlorophyll content as a result of salinity stress have also been reported for cotton 46 , pumpkin 47 and spinach 48 .
Carotenoids. ANOVA results showed that the main effect of populations, salinity and the interaction of populations on salinity were signi cant for carotenoid trait (Table 8). The effects of salinity interaction for carotenoids showed that the highest and lowest amounts were obtained in Esfahfo populations at a stress level of 9 dS / m with 0.155 mg / g and Yazd at a stress level of 9 dS / m with 0.03 mg / g, respectively (Table 3).
Protein. For malondialdehyde (MDA), the results of analysis of variance revealed that the main effect of populations, salinity and the interaction effect of salinity on populations were signi cant (Table 8). The effects of populations×salinity for MDA showed that the highest and lowest amounts were related to Qazvin and Esfahfo populations in control conditions with 5.36 and 1.46 nmol / m leaf fresh weight, respectively (Table 3).

Conclusion
Ajowan is an important medicinal plant with different food and pharmaceutical applications. In the present study, a two year morphological variation was performed to select some populations for slat stress study. So, the second part of study explored the responses of different ajowan populations to different salt treatments. As a result, Arak (C), Yazd (C), Yazd (LS), and Qazvin (SS) recorded superior EO yields, suggesting them as the best populations with acceptable salt tolerance for producing the highest amounts of favorable metabolites. Their main EO constituents were identi ed to be thymol, p-cymene, and γ-terpinene. It is interesting that different amounts of the main constituents can be usefully used in pharmaceutical and food applications.  Table 9). The study was in compliance with relevant institutional, national and international guidelines and conservation policy of endangered plants.
The seeds were sown in a randomized complete block design (RCBD) in under eld conditions at Lavark Research Farm of Isfahan University of Technology.
The soil texture of the eld was a clay loam with a bulk density of 1.4 g cm -3 and a pH of 7.8. The plot size was 1×2 m 2 and the individual plants were spaced 30 cm apart.
Salt stress experiment. In this experiment, four selected ajowan populations (namely, Yazd, Esfahfo, Qazvin, and Arak) were used. The populations were chosen based on their geographical origin, oil yield and seed weight. Fifteen seeds from each population were sown under controlled greenhouse conditions including a temperature of 25 °C and an average humidity of 50%. Each pot contained nine kilograms of soil at a soil to sand ratio of 3:1. Salt treatments were performed at early owering stage. The experiment was conducted in a factorial design with a randomized complete block design layout replicated three times. Four treatments of 0 (control), 60 (Low stress=LS), 90 (Moderate stress=MS), and 120 mM NaCl (Severe stress=SS) were applied. Prior to the owering stage, the plants were exposed to different levels of salinity supplied in normal irrigation water. For the control treatment, the pots were irrigated 2 days in the week. In each irrigation applied two liters normal water to each pot. The salt solution was gradually applied in the following steps over a period of three weeks: Initially, two liters of the saline water was applied at 60 dSm -1 to all the experimental pots during the rst week followed by a treatment of 90 dSm -1 in the second week, and one of 120 dSm -1 used as severe stress treatment during the third week. The pot soils were leached before each treatment to avoid salt aggregation. At the end of the salt treatment period, total soil electrical conductivities of all the pots, including the control, were determined using an EC meter and the values were recorded (3.1, 4.8, 6, and 8.5 dSm -1 ).
Essential oil extraction. A Clevenger type device was used to extract the essential oil from the seeds. Brie y, 10-20 g of seed was in used in every hydrodistillation to which 400 mL of distilled water was added and boiled for ve hours. Subsequently, the essential oil was collected in a glass container and the yield was reported based on dry matter.
GC/MS analysis. Essential oil compositions were determined by gas chromatography using the Agilent 7890 mass selective detector (Agilent Technologies, Palo Alto, CA, USA) with a HP-5MS column (30 m × 0.25 mm, 0.25 μm lm thickness). Oven temperature was set to 60 °C and held constant for 4 min before it was increased to 260 °C at a rate of 4 °C/min. The temperature of the GC injector port was kept at 290 °C and that of the detector at 300 °C. Helium was used as the carrier gas at a ow rate of 2 mL/min. The mass unit was operated at an ion source temperature of 240 °C and an ionization voltage of 70 eV. The RI s of the EO components were calculated experimentally using the retention time of the homologous n-alkane series (C 5 -C 24 ) 49 . The percentages of EO components were computed from the GC/MS peak areas without any correction factors.
Phenolic and avonoid evaluation. To prepare a sample extract, 100 mL of 80% methanol was added to 6 g of the seed powder samples and shaken slowly for 24 h. Then, the solution was ltered to remove the solid residues and collected for further experiments. Phenolic compounds were determined according to the Folin-Ciocalteu method 50 . Brie y, 2.5 mL of the Foline-Ciocalteu's reagent (1:10 diluted with distilled water) was mixed with 0.5 mL of the methanolic extract. The samples were incubated for 5 min at room temperature, then 2 mL of 7.5% sodium carbonate solution was added in a tube test and shaken well.
The mixture was maintained at 45 ℃ in a hot water bath for 15 min. Then, the absorbance of the mixture was measured at 765 nm using a spectrophotometer. Tannic acid equivalents (TAE) were used as the reference standard and the total phenolic content (TPC) was expressed as mg of TAE per gram of each extract on a dry basis.
The aluminum chloride colorimetric method was used to determine total avonoid content (TFC) as described by Tohidi et al. 33 . In initial, a volume of 125 µL of the extract was added to 75 µL of a 5% NaNO 2 solution. The studied samples were kept in the dark for 6 minutes before a solution of 10% Alcl 3 (150 μL) was added to each and maintained in the dark for an additional ve minutes to complete the reaction before a solution of 5% NaOH (750 µL) and water (2500 µL) were added. The absorbance of the samples was determined at 510 nm. The TFC was presented in mg of quercetin equivalents (QE) per gram of the extract.
Antioxidant capacity DPPH assay. Antioxidant activity performed using the DPPH scavenging method as described in Baharfar et al. 51 with some modi cations. Brie y, the ajowan extracts were prepared in the different concentrations of 50, 300, and 500 mg/l in methanol. At the rst, 5 mL of 0.1 mM DPPH (2, 2diphenylpicrylhydrazyl) methanol solution as the free radical source and kept for 30 min at 25 °C. The absorbance was reported at 517 nm against a blank (plant extract without DPPH) and BHT was used as standard controls for comparison. After that, by plotting the graph of extract concentrations against the scavenging activity, a speci c concentration of the sample that needed to provide 50% inhibition (IC 50 ) was calculated.
Reducing power. The reducing power of each extract was assessed using the method described in Gharibi et al. 52 . Brie y, 2.5 mL of the methanol extracts (with the different concentrations of 50, 300, and 500 mg/l) plus BHT were mixed in a solution of phosphate buffer (2.5 mL, 0.2 M, pH = 6.6) and 2.5 mL of 1% potassium ferricyanide [K 3 Fe (CN) 6 ]. The mixture was initially incubated at 50º C for 20 min before 2.5 mL of trichloroacetic acid (10%) was added and the reaction mixture was centrifuged at 3000 rpm for 10 min. Finally, the supernatant obtained after centrifugation (2.5 mL) was mixed with 2.5 mL of deionized water and 0.5 mL of ferric chloride (0.5 mL, 0.1%). The absorbance of the resulting solution was read at 700 nm versus a blank. The increased absorbance of the reaction mixture signi ed a greater reducing power.
Malondialdehyde (MDA) content. In the present study, malondialdehyde content was measured as an indicator for fatty acid peroxidation. For this purpose, at the end of the third week of salinity stress, 1 g of fresh leaf sample was powdered with liquid nitrogen and then 5 ml of 0.1% TCA was gradually added and completely homogenized. In the next step, the homogenized material was centrifuged at 10,000 rpm for 5 minutes. Then, 500 μl of the supernatant was removed and 2 ml of 20% TBA + 0.5% TCA solution was added. The samples were heated at 95 ° C in Ben Marie for 30 minutes and then rapidly placed in ice.
After cooling, they were again centrifuged at 10,000 rpm for 15 minutes. The absorbance of the supernatant was read at 532 nm.
Hydrogen peroxide (H 2 O 2 ) content. For this purpose, the samples were powdered with liquid nitrogen and then 5 ml of 0.1% TCA was gradually added to it and completely homogenized. Then, the homogenized material was centrifuged. Then 500 μl of potassium phosphate buffer (prepared from KH 2 PO 4 and K 2 HPO 4 ) was added. Finally, the absorbance at 390 nm was read by a spectrophotometer. The amount of H 2 O 2 was determined using a standard line drawn with speci ed amounts of H 2 O 2 .
Assessment of ascorbate peroxidase activity (APX). To measure the activity of ascorbate peroxidase, 250 mM phosphate buffer, 1.2 mM hydrogen peroxide, 0.5 mM ascorbic acid and 0.1 mM EDTA were mixed. Enzyme activity was initiated by adding hydrogen peroxide to the mixture. Reduction of absorption due to ascorbic acid peroxidation at 290 nm for two minutes was read. Absorption changes per minute were used to calculate enzyme activity.
Assay of guaiacol peroxidase activity (GPX). Guaiacol peroxidase activity was evaluated. The reaction medium consisted of 25 mM potassium phosphate buffer, 40 mM hydrogen peroxide and 20 mM guaiacol. The reaction was started by adding 100 μl of enzyme extract to a nal volume of 3 ml. Increased adsorption was recorded by tetragyakol formation at 470 nm for 3 minutes. The enzyme activity was then expressed as changes in absorption per minute per gram of fresh weight per minute.
Protein assay. The fresh shoots were weighed and homogenized with 2 ml of 0.1 M phosphate buffer. After homogenization, each sample was transferred to 2 ml vials. The samples were then centrifuged at 15000 rpm for 12 minutes at 4 °C. Bradford (1976) 53  Statistical analysis. The data analysis was accomplished using SAS (version 9.1). Cluster and PCA analyses were performed using Stat graphics ver. XVI.I.

Declarations
Each of authors contributed to this study as following: G.M. performed experiment and contributed to analysis and interpretation of data, and writing the manuscript. M.R. and A.A. contributed to study conception and project design and critically revised the manuscript for important intellectual content. P.Y. was responsible for data analysis. M.H.E. was responsible for a part of project design.   Table 9. Geographical location of 28 ajowan populations. Figure 1 Dendrogram generated from cluster analysis of 28 ajowan (Trachyspermum ammi L.) populations based on agro-morphological characters and essential oil content using WARD based on the Squared Euclidean dissimilarity calculated of 2017.   Evaluation of antioxidant capacity based on reducing power in ajowan extracts as compared to BHT. Control (C), 60 (Low stress=LS), 90 (Moderate stress=MS) and 120 dSm-1 (Severe stress=SS). BHT: butylated hydroxytoluene.