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 accessions12,13. 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 influence of water deficit 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 field 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 method40. 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 filter 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 flavonoids, evaluation of non-enzymatic antioxidant activity, and HPLC analysis of phenolic compounds.
Physiological characteristics
Relative Water Content
At the final stages of water stress, five 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 formula41:
% 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 fluid 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 equation43:
EL = (EC0 / EC1) * 100.
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 compound44. 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 first wavelength. In calculating the amount of malondialdehyde, extinction coefficient (155 mM/cm) was also taken into account. The amount of malondialdehyde (μmol/g fresh weight) was expressed using the following equation:
MDA = [(532 nm – 600 nm) / (QD * QF)] * DF. MDA = Malondialdehyde content in nanomoles per gram of fresh weight. QD =Cuvette diameter (1cm). QF = Extinction coefficient (155 mmol/cm). DF = Dilution factor (20).
Antioxidant enzymes activity
An exact amount of 0.25 g of crushed tissue with digital scales were weighed and transferred to the falcons and then, 2.5 ml of extraction buffer was added. All extraction procedures were performed at 4°C 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 nm46. Catalase activity (CAT) was measured at 25°C using a spectrophotometer which was set at 240 nm48. The activity of guaiacol peroxidase was measured at 470 nm as well. Ascorbate peroxidase activity was measured according to Ranieri et al (2003)48. It should be noted that the reaction between ascorbate peroxidase and ascorbic acid and H2O2 produces dehydroascorbate, which can be read at 290 nm.
Phytochemical evaluation
Total phenol content
The total phenol content was measured by Folin ciocalteu reagent49. First, 0.5 ml of the cyanoacetate reagent was mixed with 4 ml of 1 M Na2CO3 solution. After adding 0.5 ml of the solution to the plant extracts and mixing the compound thoroughly, the mixtures were incubated at room temperature for 15 minutes. Finally, the absorbance of the samples was measured by a Bio-Rad (Hercules, CA, USA) microtiter plate reader at 765 nm. All samples were analyzed in three replications and the total phenol content was calculated by standard curve of gallic acid at concentrations of 0, 50, 100, 150, 250 and 500 mg/l.
Total flavonoid content
The total flavonoid content was measured by aluminum chloride colorimetric method based on the guidelines set by Quettier -Deleu's (2000)50. For this purpose, 50 µl of the standard extract or solution, 400 µl of 2% aluminum chloride solution and then 1200 µl of 5% potassium acetate solution were mixed. After 40 min incubation at 37°C, the samples were read at 415 nm by a Bio-Rad (Hercules, CA, USA) microtiter plate reader. Three replications were applied for all samples and the standard flavonoid content of the samples was calculated by plotting standard curve at concentrations of 0, 50, 100, 150, 250 and 500 mg/l.
Antioxidant capacity
Evaluation of antioxidant capacity by DPPH method
Antioxidant properties of methanol extract of tarragon accessions were evaluated by DPPH reagent (2, 2-dipheny-l-picrylhydrazyl). In this method, the ability of the extract to trap DPPH radicals and transfer electron or radical hydrogen and convert the DPPH radical form to the reduced DPPH-H form was evaluated. Determination of DPPH radical scavenging activity was performed according to the method of Choi et al (2002)51 and the absorbance was read at 517 nm using Bio-Rad (Hercules, CA, USA) microtiter plate reader.
Evaluation of antioxidant capacity by FRAP method
The FRAP method is based on the reduction of Fe3+ -TPTZ (yellow) to Fe2+ -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 read52.
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 filtered methanol extracts were injected into the device. In order to identify the peak of each phenolic compound, the retention time in the sample was compared with the standard retention time of each injection. The type and amount of each material in the samples were determined based on the inhibition time and the area under the outlet curve and compared with the calibration curve obtained from different standard concentrations. The ultraviolet detector used was tuned at two wavelengths of 280 and 350 nm53.
Table 1. Mobile phase application injected into the HPLC device column.
Phosphoric acid (%)
|
Water (%)
|
Mobile phase velocity (ml / min)
|
Time (min)
|
Phase
|
90
|
10
|
1
|
1
|
0
|
90
|
10
|
1
|
5
|
1
|
95
|
5
|
1
|
10
|
2
|
95
|
5
|
1
|
10
|
3
|
90
|
10
|
1
|
15
|
4
|
90
|
10
|
1
|
10
|
5
|
Statistical analysis of data
Analysis of variance was performed according to the experimental design using statistical software of SAS (SAS Institute, Cary, NC, USA 1990). Comparison of mean treatments was done applying the least significant difference (LSD) at the 0.05 level of significance. Correlation of phytochemical and physiological characteristics was also performed using SPSS software 16 (SPSS Inc., Chicago, IL, USA, Norusis 1998). Excel 2013 was also applied to draw other graphs.