Two year analyses in all populations. The results of two-year analysis of variance revealed significant differences among the studied genotypes for all traits. Also, the effect of year was significant for all. The interaction of G*Y (Genotype×Year) was also significant for all the studied ones (Table 1).
During the first 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 flowering branches ranged from 6.16 (Ardebil) to 20.83 (Khormo). Arak and Farsfars population recorded the lowest inflorescence diameter, while IPK2, and Khorsar population possessed the highest inflorescence 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 inflorescence, respectively. Number of flowers 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 cm2) and the highest (1548.7 cm2) 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 flowering branch was measured in the population Ardebil, while the lowest (10.5) was measured for Hamdan (Table 2). Inflorescence 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 inflorescence, while Khorbi recorded the lowest one. Number of flowers 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 cm2 (Khorbi) to 1896.9 cm2 (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 populations10. High variation in two studied years can be resulted from environmental fluctuations as previously reported in other Apiaceae plants including ajowan11, fennel12and cumin13.
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 first year, group 1, included Khorsar, Arakkho, Yazsad, Yazd, Esfahfo, Farsmar and Ardebil populations with the highest essential oil yield, while group 2 classified 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 influenced 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). However, Esfahfo and Qazvin populations recorded increases in their EO yields.
At a low salt stress (LS), the highest (4.39%) oil yield was recorded for Yazd while Arak exhibited the lowest (3.01%) yield . A trend in EO yield similar to that observed under the control conditions was observed under the moderate stress (MS) treatment such that the Arak and Esfahfo populations recorded the highest (4.22 %) and lowest (2.64%) oil yields. Under severe stress (SS) conditions, Qazvin recorded the highest (4.26%) essential oil yield while the lowest (3.77%) belonged to Esfahfo (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 confirmed 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 populations16. 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 accumulation7. 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 populations17.
Essential oil composition. Table 4 reports all the EO constituents detected in the four studied populations under the different salt treatments. Clearly, there are high chemical polymorphisms among the Iranian ajowan populations with thymol, γ-terpinene, and p-cymene identified as the major components. It is seen in this Table that the amount of thymol ranged from 32.7±0.05% in Qazvin under the LS treatment to 54.29±0.02% in Qazvin under the control conditions. The highest (32.81±0.02%) and lowest (21.71±0.01%) amounts of γ-terpinene belonged to Qazvin (LS) and Esfahfo (SS), respectively. p-Cymene content varied from 26.16±0.02% in Esfahfo (LS) to 18.74±0.01% in Arak (C). Among the few studies reporting on the essential oil composition of Iranian ajowan populations, Moazeni et al.18 reported γ-terpinene (23.92%), p-cymene (22.9%), and thymol (50.07%) as the major compounds of the essential oil from one population collected in Kazerun, Iran. Moein et al.19 identified γ-Terpinene (48.07%), p-cymene (33.73%), and thymol (17.41%) as the major constituents of one population grown in Firoozabad, Iran. In Esfahan population, the most abundant components of the oil were reportedly thymol (44.5%), γ-terpinene (26.6%), p-cymene (21.6%), limonene (1.1%), and carvacrol (0.3%)20.
The effect of salt stress on thymol content has also been reported in the genus Thymus8. 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 well15. In the case of ajowan, seeds are harvested at full maturity and monoterpenes mostly begin to increase from the full flowering stage to seed maturity2. Moreover, salt stress reportedly affects the biosynthesis of isoprenoids as a result of its influence on isoprene subunits.
The different trends observed in thymol accumulation in the populations examined in the present study make it difficult to draw definitive 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 compounds4. 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 filling21. Finally, the radical scavenging mechanism has also been suggested for changes in metabolites during stress conditions4,16. 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 influenced by the salt stress treatments. Salt stress caused a significant reduction in seed yield per plant of T. ammi (Table 3). A maximum reduction in seed yield was observed at the (LS) treatment. Number of seeds per plant varied from 47.66 to 68.75 g under the control condition, the lowest and highest recorded for Arak and Yazd, respectively (Table 4). At a low salt stress (LS), the highest (67.34) Number of seeds per plant was recorded for Esfahfo while Arak exhibited the lowest (40.46) number of seeds. Under the moderate stress (MS) treatment observed that the Esfahfo and Qazvin populations recorded the highest (49.50) and lowest (29.22) number of seeds. Under severe stress (SS) conditions, Arak recorded the highest (34.10) number of seeds per plant while the lowest (28.59) belonged to Qazvin (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. alfalfa22,23, carrot24, and cumin25.
Total phenolic and flavonoid 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 conditions26. In plants exposed to abiotic stresses, the rate of cellular oxidative damage can be controlled by the plant's capacity to produce antioxidants27. 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 broccoli28. In response to salt stress, whereas NaCl treatment elevated TPC levels in maize29 and red pepper30.
Similarly, the ajowan populations studied exhibited substantial differences with regard to their flavonoid content. While Yazd (MS) recorded the highest TFC content (5.94 mg QE g-1DW), Yazd (C) exhibited the lowest (3.48 mg QE g-1 DW). Moreover, a significant 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 flavonoids among their subcellular sections. Metabolically, plant polyphenols, such as flavonoids and phenolics, are biosynthesized through several pathways4. The underlying mechanism involved in flavonoid 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 condition27. It is thought that moderate salinity stress induces the normal saline tolerance pathway via increasing flavonoid contents30. 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 flavonoids 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 IC50 values were found to vary from 1566.985 µg/mL to 5889.99 µg/mL. More specifically, 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 IC50 observed among different species might be interpreted with recourse to the diversity in their polyphenolic components14. 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 carvacrol31. 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 species32. 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 reported a high variation in the antioxidant activity of some Indian Apiaceae spices based on their DPPH assay results. Similarly, Akbarian et al.34 used the DPPH method to observe a high variation in the antioxidant capacity of three Ferula species.
Reducing power. The reducing power of the studied ajowan populations was found to rise with increasing EO concentration (Figure. 4). Clearly, the highest antioxidant capacity in absorbanceat 700 nm was obtained in Yazd (SS) (0.84) in 500 mg/l while Arak (C) exhibited a lower activity than BHT. From among the ajowan populations, in absorbanceat 700 nm Yazd (SS), Yazd (MS), Esfahfo (SS) and Yazd (LS), recorded reducing powers of 0.84, 0.69, 0.44, 0.39, and 0.39, respectively, which were higher than those recorded for the other populations (Figure. 4). Similarly, Afshari et al.35 reported that strongest reducing power was observed in A. pachycephala at concentrations of 300 and 500 mg/l. In a similar study based on the reducing power model, Vafadar Shoshtari et al.6 found that myrtle subjected to salt stress showed elevating antioxidant activity with increasing extract concentration.
Cluster and principal component (PCA) analysis. Cluster analysis was performed using the main essential oil components, TPC, TFC to detect any similarities among the ajowan populations studied. The results are illustrated in the dendrogram in Figure 5. The analysis provided further information regarding the distribution of ajowan populations in terms of their essential oil yields and suggested diversified chemical compositions as a result of the different salt stress conditions investigated. The results obtained grouped the ajowan populations into the following four clusters. The first consisted of the two accessions of Qazvin (C) and Arak (LS). The first group consisted of the two accessions of Qazvin (C) and Arak (LS) both rich in thymol (54.29±0.02, 50.05±0.05). The second group was further divided into two subgroups. The first consisted of Qazvin (MS), Arak (MS), Yazd (LS) and Esfahfo (MS). The main components of which were TFC (5.35, 4.37) and TPC (147, 125.99). Finally, Yazd (MS) and Esfahfo (SS) with higher quantities of p-cymene (20.55, 19.21) was assigned to the second subgroup. The third group consisted of Esfahfo (C), Arak (SS), Yazd (SS) and Arak (C). The main components of this group were high quantities of essential oil yield (4.77, 3.22). Fourth group consisted of Qazvin (LS), Qazvin (SS),Yazd (C) and Esfahfo (LS) the main components of which were γ-terpinene (32.81, 22.75) (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 stress21.
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 first and second components explained 69.32% of all the variation observed (Table 5) while the first component showed 44.40% of the total variation. Finally, PC2 explained 24.91% of the total variance. The first 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 compounds36. 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 (-0.961*), and DPPH and γ-terpinene (-0.990**). Under Low salt stress (LS), positive correlation was observed between DPPH and p-cymene (0.947*). The result of correlation analysis at moderate stress (MS) conditions exhibited the negative correlations thymol and γ-terpinene (-0.994**), while the correlation between total phenolic and thymol (0.970*), and reducing power and total phenolic (0.969*) was positive. Finally, at severe stress (SS) environment, a positive 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 pathway33. Previous reports have shown different trends in the accumulation of these components in thyme leaves33. 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 confirmed by the results reported elsewhere that highlighted changes in monoterpene frequencies based on phenological differences and harvested organs37. Finally, the positive correlations between the precursors and final products might be attributed to the complete transformation of the precursors.
Physiological evaluations
Malondialdehyde (MDA) and hydrogen peroxide (H2O2). 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 significant (Table 8). The effects of populations× salinity for MDA showed that the highest and the 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 8). It has been reported that leaf MDA content at 6 dS / m NaCl level has significantly increased in different Sesamum indicum cultivars as compared with control38. Unsaturated fatty acids are the main constituents of membrane lipids that are prone to peroxidation by free radicals due to salinity stress39. In this regard, MDA content is an indicative of oxidative damage40.
A significant difference in hydrogen peroxide (H2O2) content was observed among the populations in the control environment and salinity stress treatments. H2O2 was decreased at salinity levels of 6 dS / m salinity level. The results of population×salinity showed that the highest and lowest values of this trait were related to Qazvin populations in control conditions with 2.40 mmol / g and Arak at 6 dS / m stress level with 0.45 mmol / g (Table 3). Hydrogen peroxide (H2O2) is an active signal molecule and its accumulation leads to a wide range of plant responses to environmental stresses, as these reactions are interdependent41. 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 cells42. 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 stress43. 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.44and Foeniculum vulgare Mill45.
Antioxidant enzymes activity. The results of statistical analysis showed that there is a significant 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 8). Interaction of salinity effects in populations for guaiacol peroxidase enzyme revealed that the highest and lowest values of this trait belongs to Qazvin populations at a stress level of 12 dS / m with 0.277 FW U mg-1 and Isfahanfo under 12 dS / m level with 0.012 FW U mg-1 was (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 significant 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 cotton46, pumpkin47and spinach48.
Carotenoids. ANOVA results showed that the main effect of populations, salinity and the interaction of populations on salinity were significant 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 significant (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).