Screening of Iranian Tarragon Accessions Based on Physiological and Phytochemical Responses Under Water Decit: Applications for Development and Breeding

In this study, screening of tarragon accessions based on physiological and phytochemical traits was investigated under water decit. The compounding impacts of water decit * accessions signicantly altered the chlorophyll and carotenoid contetnt, malondialdehyde, electrolyte leakage, superoxide dismutase, catalase and ascorbate peroxidase. The HPLC analyse revealed the presence of chlorogenic, syringic, ferulic, vanilic, chicoric and p-coumaric acids as major phenolic acids, while quercetin and herniarin were detected as the predominant avonoid and coumarin compounds in the extracts. The quality and quantity of tarragon secondary metabolites were impacted by water decit, suggesting that drought stress either increased the amounts of some common compounds or introduced some new compounds that were not present under normal conditions. In some genotypes, the content of some secondary metabolites in tarragon had the highest values under severe water decit stress. The results indicated that Hamadan, Varamin and Estahbanat accessions could be introduced as tolerant accessions. Due to the very different response of tarragon accessions to water decit and the existence of diversity between these accessions, the ndings of the present study could be an effective step in identifying and achieving homogeneous, drought-tolerant and high-yield potential accessions, and may help tarragon breeding programs and development of cultivation.


Introduction
A severe decrease in water availability most likely leads to drought stress and some unsatisfactory physiological and phytochemical changes in plants. Drought is the most common environmental stress and is considered as an important factor limiting crop production worldwide which decreases the production up to approximately 30-40% of the global agricultural elds 1 .
The herbaceous and perennial Artemisiadracunculus, which belongs to the Asteraceae family, subfamily of Radia, and Artemisia genus, has woody and yellowish-green or brownish-green stalks of 30 to 150 cm in height, depending on the climate of its growth environment. It is one of the 20 most commonly grown herbs in Europe and is consumed as fresh, dry and frozen products 2 . The main classes of secondary bioactive metabolites of tarragon include coumarins, avonoids and phenolic acids 3 . Previous researches mostly focused on identi cation of essential oil compounds of tarragon and their diversity 4,5 . Some studies, however, have investigated the tarragon's polyacetylene derivatives, coumarins, avonoids and, to a lesser extent, the sesquiterpene, vitamins, tannins and alkaloids found in its aerial parts.
Identifying and screening of plant germplasm tolerant to water de cit conditions would be the underlying objectives of the relevant scienti c researches. Wild germplasms and native genotypes are valuable genetic resources for crucial physiological traits such as drought tolerance that could be identi ed and applied in breeding programs 6 . Obviously, among the different populations of a plant species, those that are more tolerant to water stress are the better choices for arid and semi-arid areas 7 . The impact of water scarcity on plant yields and adverse changes of active substances of medicinal plants must be thoroughly evaluated. Any attempt to genetically modify drought tolerance using the existing genetic diversity largely requires an e cient screening or other functional approaches to be rapid and capable of evaluating plants at sensitive growth stages 8 .
Populations of an individual species of medicinal plants that grow in different ecological conditions constitute inconsistent types in terms of quantity and quality of their active ingredients, which, in turn, leads to dissimilarities in the range of their medicinal and biological activities. Genetic exibility of plant populations allows the emergence of the diversity and gradually forms populations of the same species in dissimilar geographic regions, which largely differ in terms of developmental, physiological, chemical and botanical activities 9 . For the reasons mentioned, native populations of medicinal plants are heterogeneous based on morpho-phenological and chemical properties 10 . Therefore, if a medicinal plant is used in industry, homogeneous cultivars with an optimal level of active ingredient should be applied in order to meet WHO objectives for optimum cultivation of medicinal plants and production of safe, stable and e cient raw materials 11 .
Even though native tarragon accessions of Iran have already been collected and some of their qualitative and quantitative characteristics have been studied 12,13 , accurate assessing their tolerance to environmental stresses, especially drought stress, has not been conducted. Obviously, some of these accessions, aside from their higher potential for cultivation, might be used in breeding programs as sources of stress resistance. This may require the relationship between drought tolerance and their physiological, morphological and phytochemical markers to be well examined. Accordingly, the present study could be a starting point for extensive scienti c researches on tarragon accessions to screen and introduce drought-tolerant cultivars in the future.
Given that Iran has the high rank in the world in terms of cultivation and production of this crop, considerable variation has been observed in the tarragon accessions in the country 12,13 . The core objectives of this study were screening twelve accessions of Iranian tarragon based on important physiological and biochemical traits relating to water-de cit tolerance and then evaluating their secondary metabolite changes under water de cit.

Variance analysis of physiological traits
The results of variance analysis of physiological traits showed that water de cit stress had a signi cant effect on measured traits (Supplementary Table 1). Signi cant differences were observed between different accessions of tarragon for all studied traits as well (Supplementary Table 2). In addition, the interactive effects of water de cit * accessions were signi cant for all studied traits except relative water content (Supplementary Table 3).

Comparison of the mean physiological traits
The relative water content of leaf under water de cit was signi cantly reduced that way the highest relative water content (74.55%) was obtained under control condition and the lowest one (62.33%) was recorded under severe stress ( Fig. 1 and 2). There was also a signi cant difference in terms of relative water content between different tarragon accessions (Supplementary Table 1). Varamin, Hamadan and Yazd accessions had the highest relative water content; whereas the lowest relative water content belonged to Isfahan, Abadeh and Semirom (Supplementary Table 2). Examining the interaction between water de cit * accessions revealed that Hamadan accession had the highest content of chlorophyll a, chlorophyll b and total chlorophyll under normal and mild stress conditions; whereas, Isfahan, Abadeh and Semirom accessions had the lowest amount of these traits under severe stress conditions. Hamadan accession had the highest content of carotenoid, while the lowest carotenoid content recorded for Abadeh, Semirom and Zarand accessions under drought stress. In general, decrease in chlorophyll a, b and total chlorophyll content was observed under water de cit in most tarragon accessions, although the rate of decrease varied greatly relating to the drought severity (Supplementary Table 3). Most of the accessions studied did not show a signi cant decrease in chlorophyll content under mild stress. In addition, when it comes to Yazd accession, there was no decrease in chlorophyll content even in severe stress treatments (Supplementary Table 3).
Despite falling carotenoid content under water de cit in most of the tarragon accessions, this valuable substance increased in Yazd, Varamin and Hamadan accessions under water de cit. Carotenoid/chlorophyll ratio signi cantly rose in all tarragon accessions under severe drought stress; whereas most of the accessions (except Zarand) did not increase under mild stress (Supplementary Table 2). Examining the interaction effects of water de cit * accessions demonstrated that Varamin and Yazd accessions had the highest activity of superoxide dismutase (17.61 and 17.32 μmol min −1 mg −1 protein, respectively), catalase (3.828 and 3.86 μmol min −1 mg −1 protein, respectively), guaiacol peroxidase (0.4853 and 0.4307 μmol min −1 mg −1 protein, respectively) and ascorbate peroxidase (3.016 and 2.957 μmol min −1 mg −1 protein, respectively) under severe stress. The ascorbate peroxidase activity of Neishabour was also considerable under severe stress (2.583 μmol min −1 mg −1 protein). The lowest activity of superoxide dismutase was observed in Semirom, Abadeh, Kermanshah and Birjand accessions under normal conditions. Semirom, Isfahan, Kermanshah and Birjand also showed the lowest catalase activity under control conditions. Semirom, Isfahan and Zarand accessions had the lowest activity of ascorbate peroxidase under normal conditions. The trend of changes in antioxidant enzymes activity in all tarragon accessions under water de cit was incremental. However, the intensity of activity changes in various accessions was somewhat different and in general, the activity of all enzymes was higher in Yazd, Varamin and Hamadan in relation to other studied accessions (Supplementary Table 2 and 3).
The malondialdehyde content and electrolyte leakage percentage of the studied accessions also increased under water de cit. Among the investigated accessions, the highest values of these traits were assigned to Semirom, Abadeh and Isfahan. In general, the highest and lowest content of malondialdehyde (14.8 and 3.453 nmol g −1 fw, respectively) were obtained in Semirom under severe stress and Isfahan under control conditions. The maximum and minimum percentage of electrolyte leakage (58.45% and 20.23%, respectively) under severe stress and control conditions was recorded for Abadeh accession (Supplementary Table 2 and 3).

Antioxidant capacity
The results showed that water de cit had a signi cant impact on antioxidant activities calculated through DPPH and FRAP tests (Supplementary Table 4). Various tarragon accessions also showed signi cant differences in terms of antioxidant capacity in both tests (Supplementary Table 5). However, in none of the applied methods, the interactive effect of water de cit * accessions on the antioxidant capacity of the extract was signi cant (Supplementary Table 6). Based on the results of mean comparison, it was observed that in DPPH assay, IC50 methanol extract of tarragon declined under water de cit.
The highest and the lowest antioxidant capacity were observed under severe stress (IC50 = 0.076) and control treatment (IC50 = 0.082), respectively (Fig. 3). The highest and the lowest antioxidant capacity (616.16 μmol of iron per gram of dry weight and 574.9 μmol of iron per gram of dry weight, respectively) were obtained under severe stress and control conditions as well (Fig. 3). In DPPH test, Birjand accession had the highest antioxidant capacity (IC50 = 0.050) followed by Hamedan (IC50 = 0.056). The lowest antioxidant capacity also belonged to Varamin accession (IC50 = 0.103) followed by Abadea (IC50 = 0.102) (S5). When performing FRAP test, Birjand, Estahbanat and Hamedan accessions had the highest antioxidant capacity (709.20, 690.80 and 686.00 μmol iron per gram of dry weight, respectively), while the lowest (402.30 μmol iron per gram of dry weight) belonged to Varamin accession (Supplementary Table  6).

Total phenol and avonoid contents
The results of variance analysis revealed that total phenolic and avonoid contents of tarragon extract were signi cantly affected by water de cit (Supplementary Table 4). Signi cant differences were observed among all of the tarragon accessions in terms of total phenolic and avonoids (Supplementary Table 5). The interactive effects of water de cit * accessions were also signi cant when total phenol and avonoid contents of the extract are concerned (Supplementary Table 6). The total phenol content of different tarragon accessions was impacted by water de cit in contrasting ways. Although there was an increase in total phenolic under water de cit in most accessions that was signi cant only in Abadeh and Yazd accessions. The interactive effects of water de cit * accessions on the total avonoids were also very similar to the total phenolic. The amount of total avonoid rose in the most accessions under water de cit, but the difference between various levels of stress was signi cant only for Abadeh and Yazd accessions (Supplementary Table 6).
Finally, the highest total phenolic content (79.81 mg gallic acid g -1 fresh weight) was observed in Abadeh accession under severe stress. The highest total avonoid (50.76 mg /g fresh weight) was obtained in Birjand accession under mild stress. Abadeh accession has the lowest total phenols and avonoids (35.64 mg gallic acid g -1 fresh weight and 21.15 mg /g fresh weight, respectively) under normal conditions (Supplementary Table 6).
Analysis of phenolic compounds of the extract HPLC analysis of methanolic extract of tarragon resulted in qualitative and quantitative identi cation of 14 phenolic compounds from three different groups. Chlorogenic acid, syringic acid, chicoric acid, caffeic acid, vanillic acid, ferulic acid, gallic acid and p-coumaric acid were identi ed and measured as major plant phenolic acids. Qualitative and quantitative evaluation of important plant avonoids including luteolin, quercetin, naringenin and apigenin were performed as well. Coumarin and herniarin were also the coumarin compounds identi ed and measured in tarragon accessions ( Fig. 3 and 4).
Variance analysis of phenolic compounds of tarragon extract showed that drought stress had a signi cant in uence on all these compounds (Supplementary Table 4). Signi cant differences were observed between the different accessions of tarragon for the detected phenolic compounds (Supplementary Table 5). The interactive effects of stress * accessions were also signi cant for all the phenolic compounds (Supplementary Table 6). whereas the lowest syringic acid was (zero) recorded for Semirom accession (Supplementary Table 6).
The highest and lowest amounts of caffeic acid (25.39 and 4.00 mg g −1 dry weight, respectively) belonged to Hamadan and Varamin accessions under normal conditions, respectively. Even though the amount of vanillic acid and ferulic acid in the extracts of most of the accessions was considerable, these compounds were not found in some accessions. The highest amount of ferulic acid and vanillic acid (242.20 and 26.71 mg g −1 dry weight, respectively) were obtained in Varamin and Estabhanat accessions under normal conditions, respectively. Chicoric acid was also observed only in Zarand, Birjand and Kermanshah accessions in low amounts (Supplementary  Table 6). Naringenin was observed only in Neyshabour and Estahbanat accessions, quercetin was found only in Isfahan, Neyshabour, Birjand, and Hamedan accessions, and apigenin was detected just in Semirom and Zarand accessions.

Discussion
According to the results of this study, various tarragon accessions had signi cant differences in relative water content of leaves (Supplementary Table 1 and 2). Comparison of relative water content showed that the accessions of Varamin, Hamadan and Yazd with the highest relative water content were probably more drought-tolerant than other accessions. The results of tarragon leaf pigment measurements showed that under severe drought stress chlorophyll a, b and total chlorophyll content decreased in all accessions (Supplementary Table 2 and 3). The loss of leaf water not only impedes chlorophyll synthesis but also appears to cause chlorophyll degradation. Drought causes the chloroplast to break down and reduce the concentration of chlorophyll. Since both chlorophyll and proline are synthesized from a common precursor called glutamate, it could be said that increased proline synthesis under drought stress leads to a decrease in chlorophyll synthesis14. The ndings of this study suggest that different tarragon accessions somewhat vary in this respect and may exhibit different degrees of drought tolerance. In this study, chlorophyll content at mild stress did not change compared to the control treatment and severe stress lowered the content of this pigment ( Fig. 1 and 2). Decreases in chlorophyll a, b and total chlorophyll content have been reported in several studies15,16. Miao et al (2020) found that chlorophyll a, b and total chlorophyll contents fell under extreme stress conditions, while the ratio of chlorophyll/carotenoid and chlorophyll a/b rose17. Our ndings demonstrated that carotenoid content of tarragon accessions had different responses to drought stress, which may qualify this trait to be considered as the main criterion for distinguishing the resistant accessions (Supplementary Table 2 and  3). In the present study, the activity of antioxidant enzymes activities increased in all accessions under drought stress, albeit with varying intensity. It seems that high intensity of antioxidant enzymes activity in Varamin, Hamadan and Yazd accessions re ect a high tolerance to drought stress. Hosseinpour et al (2020), reported that the activity of superoxide dismutase, ascorbate peroxidase, polyphenol oxidase catalase, guaiacol peroxidase and total protein and proline contents rose as a defense mechanism in Echinacea purpurea under drought stress conditions18. Signi cant increase in peroxidation of cell membranes in leaves under drought stress could be due to increased production of oxygen free radicals under stress conditions that cause their reaction with fatty acids of cell membranes and their oxidation19. The ndings of the present study showed that the content of malondialdehyde and electrolyte leakage (%) increased in all studied accessions under drought stress (Supplementary Table 3). However, in relation to the other accessions, Semirom, Isfahan and Abadeh contained higher contents of malondialdehyde and electrolyte leakage, which may indicate their higher susceptibility to this abiotic stress. Numerous reports have shown that lipid peroxidation is more prevalent in drought-sensitive species and genotypes than resistant stress20. Mohasseli et al (2020) found that the chlorophyll and relative water content decreased steadily under drought stress. Malondialdehyde and electrolyte leakage (%), and H2O2 content signi cantly increased in Melissao cinalis as well21. Their results indicated that drought stress enhanced proline content and antioxidant enzymes, including catalase, ascorbate peroxidase and polyphenol oxidase activities. Identi cation of phenolic components of methanol extract by high performance liquid chromatography indicated the presence of chlorogenic, syringic, chicuric, caffeic, vanillic, ferrulic and p-coumaric acids, and luteolin, quercetin, naringenin, apigenin, herniarin and coumarin ( Fig. 3 and 4). Numerous studies have been carried out on the quantitative and qualitative identi cation of phenolic compounds of tarragon aerial parts3,22,23,24. In current study, the results of the free radical scavenging activity of tarragon extract using the methods of DPPH and FRAP showed the high antioxidant capacity of the plant extracts ( Fig. 3 and 4). In addition, two different antioxidant capacity assays showed similar results indicating some differences in antioxidant capacity of various accessions ( Fig. 3 and 4). Signi cant correlation between total phenol content and total avonoid content with antioxidant capacity of the extract in both methods implies the important role of polyphenolic compounds in the antioxidant capacity of the extract (Supplementary Table 7). Positive correlations between polyphenols and antioxidant capacity of the extract have been reported in several studies25,26. The results of Bettaieb et al (2011), showed the scavenging activity IC50 and reducing power EC50 increased at moderate and decreased at severe stress. Liu et al (2011) found that moderate and severe drought stress substantially reduced root and shoot dry weight, while the root /shoot ratio rose. In addition, with increasing drought, the percentage of salvianolic acid B, dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone IIA increased signi cantly and the percentage of rosmarinic acid decreased27. Their ndings implied that the moderate level of drought may act as a suitable stimulus to increase the amount of secondary plant metabolites. In order to improve the quantity of active compounds and to homogenize them, superior plant varieties with the same genetic background should be developed28. The results of this study revealed that proper application of active compounds in the extract should be concerned as an important part of the breeding purposes. Among the tarragon accessions, there was a proper diversity along with high biological activity. Nowadays one of the most chief concerns regarding the cultivation of medicinal plants is the changes in quality and quantity of these plants under various environmental conditions. In the meantime, water de cit is one of the major problems in crop production in arid and semi-arid regions, including Iran9,29. Ghahremani et al (2020), observed that the total phenolic content in leaf and ower, phenolic acid, avonol and quercetin in ower of Verbascum songaricumSchrenk increased as a defense mechanism under drought stress conditions30. Drought stress is generally considered as a limiting factor in agriculture and one of the main causes of crop yield decline. However, medicinal plants cultivated in semi-arid Mediterranean regions usually produce more biologically active compounds than similar plants in moist regions. The distribution of essential oil-rich aromatic plants is higher in arid regions compared to other climatic areas.
Therefore, secondary metabolites are likely to be effective in drought tolerance mechanisms by reducing transpiration. Although other factors might contribute to elevate secondary compounds in semiarid regions, the main cause of these metabolites increases is due to the differences in the amount of water available to the plant. Because the reactions that the plant exhibits in response to stress affect the entire metabolism to a large extent, the production and accumulation of secondary metabolites are also impacted31,32. Kleinwächter and Selmar (2015) have shown that water scarcity combined with high light intensity resulted in stomatal closure, thereby reducing carbon dioxide absorption and stabilization31. In this study, total phenol, total avonoid and antioxidant the capacity of methanolic extract of tarragon increased following drought stress ( Fig. 3 and 4). Given the effective role of polyphenols in protecting the plant against oxidative stress induced by drought, increasing the amount of these compounds in the plant under drought stress seems likely. Increased synthesis of secondary metabolites under drought stress has been reported in several studies16,27,33. There is ample evidence of increasing levels of all secondary metabolites, comprising simple and complex phenols, terpenes, alkaloids, glycosides, etc., following drought stress. There is, arguably, no doubt about the increase in natural compounds under drought, but the plant growth and drug yield could be the primary cause of the decline in metabolic Their results implied that water de cit stress reduced all morphological traits, while H2O2 and malondialdehyde content increased signi cantly under water de cit stress. It was also observed that water de cit stress had a signi cant effect on the content of polyphenol oxidase and superoxide dismutase. They found that total phenol and avonoid contents as well as the free antioxidant activity signi cantly decreased as well. They conclude that moderate stress increases the biochemical properties of peppermint and ultimately enhances the plant resistance36. Similarly, in this study, the content of abscisic acid in the treated plants increased. However, increasing levels of total phenol and total avonoids due to drought stress have been reported in many studies20,36. Investigation of changes in phenolic composition of extracts revealed that the amount of herniarin and luteolin rose in most tarragon accessions impacted by water de cit (Supplementary Table 5 and 6). However, the magnitude of this increase in different accessions was affected by stress so that the difference between various levels of stress in some accessions was not signi cant. For example, the increase of herniarin in Hamadan, Kermanshah, Semirom, Abadeh, and Anonymous was more than other accessions. The trend of increasing the amount of luteolin in Kermanshah accession was more obvious in relation to the other studied accessions. In contrast, stress-induced changes in chlorogenic acid content in many tarragon accessions followed a falling pattern. The most severe decreases in chlorogenic acid were observed in Semirom, Abadeh and Estahbanat accessions, while no signi cant changes were observed in Varamin, Yazd, Birjand and anonymous accessions. The trend of caffeic acid changes under drought stress was increasing in Neyshabour, Estahbanat, and Anonymous accessions, but in Hamedan, Kermanshah, Isfahan, Abadeh and Zarand accessions followed a decreasing pattern (Supplementary Table 5 and 6). Ritesh et al. (2014) found that the amount of artemisinin, artemisinic acid, dihydroartemisinic acid and artemisia signi cantly fell under drought stress37. The results of Bettaieb et al (2011) showed that the reactions of avonoids content, cinnamic acids, and benzoic acids were inconsistent at different drought levels. The content of cafeic acid, vanillic acid, rosmarinic acid, p-coumaric acid, cinnamic acid, dihydroxybenzoic acid, ferulic acid, trans-cinnamic acid, quercetin 3-D-galactoside, quercetin, campherol, naringin, apigenin, amento avone and avone rose at moderate levels of drought stress. With the increase of stress intensity, however, the levels of these compounds decreased, but the content of chlorogenic acid, trans-2-Hydroxycinnamic acid and catechin increased. Meanwhile, scavenging activity of IC50 and reducing power of EC50 also rose at moderate intensity, but fell at severe stress33. Since Varamin and Hamadan accessions had almost the highest values in terms of the most phytochemical traits, this part of the results con rmed the ndings of the rst and second parts of the experiment. Interestingly, Kermanshah, Hamadan and Varamin accessions, which were selected as the most tolerante accessions according to the results of three different sections, did not have apigenin and naringenin. The question here is whether there is a relationship between the two compounds and drought tolerance or sensitivity. Mentioning this theory according to the results of this research is only a speculation which needs to be con rmed by detailed and coherent research. Meanwhile, in this study, the type and quantity of tarragon secondary metabolites were affected by water de cit. The effect of drought stress is like a double-edged sword, because on the one hand it reduces plant dry matter and on the other hand, it increases the amount of secondary metabolites. In fact, an increase in the amount of metabolites offsets the decrease in dry yield, so drought stress can not be used as a suitable stimulus to increase the secondary metabolites of tarragon, unless the tolerant cultivars are available38,39. Due to the tolerance of Hamadan, Varamin and Kermanshah to drought stress, it seems that drought stress could be applied as a suitable stimulus to increase the amount of tarragon secondary metabolites or that of these accessions as suitable parents take advantage in the classic breeding programs and biotechnology approaches. Interestingly, the measured traits in some genotypes did not change signi cantly under mild stress compared to normal conditions, candidating these cultivars as suitable ones for cultivation in areas that naturally suffer from mild water de cit stress. In addition, the quality and quantity of secondary metabolites were signi cantly impacted by water de cit stress, as the content of some of the measured compounds substantially increased under mild stress. The content of phenol, avonoid, herniarin, luteolin, apigenin, gallic acid and quercetin reached their highest values under severe stress conditions. Accordingly, when tarragon cultivation is done only aimed at producing secondary metabolites with different purposes, like medicine and pharmacy, cosmetics and so on, some of these genotypes appear to be suitable candidates in areas with water shortages.

Method
Tarragon accessions cultivation and applying treatments The plant materials used in this study were obtained from the collection of Research Medicinal Plants Institute of Tehran Shahid Beheshti University. It is noteworthy that the evaluated accessions were previously selected from 26 populations collected from the main tarragon cultivation areas in Iran and according to traits such as vegetative yield, essential oil yield, morphological and phytochemical characteristics and their genetic diversity, were selected as desirable accessions 12,13 . The use of these accessions were carried out in accordance with relevant guidelines and regulation. The farm soil was analyzed before planting tarragon. The results of soil analysis and climatic conditions of tarragon cultivation area are presented in supplementary Table 8 and 9, respectively. In this study, the in uence of water de cit on 12 accessions of tarragon accessions was evaluated in a split plot design by randomized complete block with three replications. Soil moisture treatments, comprising irrigation at 100 ± 5%, 80 ± 5% and 60 ± 5% of eld capacity, as main-plots and tarragon accessions as sub-plots were considered.

Sampling and physiological evaluation
The leaves of plants were harvested for evaluation of physiological traits, and the samples were kept in the freezer (-80°C) until measurement.

Plants harvesting and extraction
The extract of the plant was provided using maceration method 40 . For this purpose, 250 ml Erlens containing 10 gr of milled dried plant, to which 100 ml of methanol-water solvent (80%) was added, were placed on the shaker for 72 hours at room temperature. The contents of the Erlens were then passed through a lter paper and the methanol solution was transferred to a rotary vacuum apparatus in order to remove the methanol from the extract. Then, the pure extracts were left in dark glass at 4°C until analysis. The extract was analyzed for total phenolic content, total avonoids, evaluation of non-enzymatic antioxidant activity, and HPLC analysis of phenolic compounds.

Relative Water Content
At the nal stages of water stress, ve leaves were selected from each plant and their fresh weight was determined. In order to identify the leaf weight in the turgor state, leaf fragments were exposed to the low light intensity at 4°C for 24 hours in distilled water, aimed to absorb the leaf cells into the turgor state. Then, the swollen parts were carefully weighed again. After that, the leaves were dried at 75°C for 24 hours and their dry weights were measured and the relative leaf water content (in percent) was obtained using the following formula 41 : % RWC = [(Wf -Wd) / (Wt -Wd)] *100.
In this formula, Wf is fresh leaf weight, Wt is swollen leaf weight and Wd is leaf dry weight.

Chlorophyll and Carotenoid contents
The content of different pigments, including chlorophyll a, b, total chlorophyll and carotenoid contents were measured according to the method reported by Şükran et al (1998) 42 . For this purpose, 0.125 g fresh leaf tissue with 10 ml of 80% acetone and 0.1 g calcium carbonate (to neutralize the acidic state of the intracellular uid and prevent chlorophyll degradation) were crushed in a mortar. After centrifugation of the extract (10,000 rpm for 10 minutes), the supernatant was applied to determine the pigment contents. Finally, the absorb light at wavelengths of 663 nm (maximum chlorophyll a absorption), 645 nm (maximum light absorption of chlorophyll b) and 470 nm (maximum light absorption of carotenoids) were read using a UV-vis spectrophotometer (UV-1800; Shimadzu Corporation, Kyoto, Japan).

Electrolyte leakage
Ten punched leaves were mixed with 10 ml distilled water. The containers were then shaken on the shaker for 24 hours at 150 rpm and electrolyte conductivity (EC0) was read. The solution containing the samples was then autoclaved at 120°C for 20 min and electrolyte conductivity (EC1) was read again after cooling. Finally, the percentage of leaf electrolytes leakage (EL) was calculated by the following equation 43 :

Malondialdehyde content
Membrane lipid peroxidation was measured based on the concentration of malondialdehyde produced by damage to the membrane and its reaction with thiobarbituric acid, which forms a colored compound 44 . The absorbance of the mixture was measured by a UV-vis spectrophotometer (UV-1800; Shimadzu Corporation, Kyoto, Japan) at two wavelengths of 532 nm and 600 nm. It is worth mentioning that, the absorption at the second wavelength is the absorption of impure fats which should be less than the absorption at the rst wavelength. In calculating the amount of malondialdehyde, extinction coe cient (155 mM/cm) was also taken into account. The amount of malondialdehyde (μmol/g fresh weight) was expressed using the following equation: in ice. After two minutes of vortex, the samples were centrifuged for 15 minutes at 4°C with 13000 rpm. That extract can be used to measure the activity of catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), superoxide dismutase (SOD) and soluble proteins. Total protein was measured according to Bradford (1976) 45 . The activity of superoxide dismutase was measured spectrophotometrically and based on its inhibitory ability to photochemical reduction of nitrobutetrazolium (NBT) at a wavelength of 560 nm 46 . Catalase activity (CAT) was measured at 25°C using a spectrophotometer which was set at 240 nm 48 . The activity of guaiacol peroxidase was measured at 470 nm as well. Ascorbate peroxidase activity was measured according to Ranieri  The FRAP method is based on the reduction of Fe 3+ -TPTZ (yellow) to Fe 2+ -TPTZ (blue) at low pH values.
For this purpose, 180 µl of FRAP solution was added to 20 µl of methanol extract and kept at 37°C for eight minutes. The absorbance of the solutions at 593 nm was read by a Bio-Rad (Hercules, CA, USA) microtiter plate reader. The blank sample containing FRAP solution was also read. Besides, Fe2SO4.7H2O solution was prepared at 0, 25, 50, 100, 150, 250 and 500 μg /l concentrations to draw the standard curve and their corresponding numbers were read 52 .

HPLC analysis
In this study, dried tarragon extract was dissolved in methanol and analyzed by HPLC Perkin Elmer series 200 Q/410 manufactured in the United States. The HPLC instrument had a quad-core 200-Q410 LCD pump, an auto-sampler and a diode array UV spectrometer. The column used was phenyl 6-carbon reverse phase with a length of 25 cm, an inner diameter of 4.6 mm and a particle diameter of 5 μm. The mobile phase consisted of 20 mM water and phosphoric acid, which entered the column in different proportions over 70 min (Table 1). 10 µl of ltered methanol extracts were injected into the device. In