Effects of drought stress on biochemical traits and its relationship with growth stage in milk thistle (Silybum Marianum L.)

Drought stress is the most critical factor limiting agricultural and medicinal plants' performance in arid and semi-arid areas. Silybum Marianum L. is a medicinal plant with antioxidant properties. In addition to the plant's genetic nature, flavonolignan production and accumulation are affected by various environmental conditions. The accumulation of secondary metabolites under drought stress was studied concerning the antioxidant defense system at the biochemical level. The purpose is to evaluate the secondary metabolites of milk thistle under non-stress conditions and different levels of drought stress and different growth conditions, as well as to identify the best level of moisture stress and the time of harvesting the plant to increase the effective compounds. Milk thistle seeds were disinfected and transferred to a Petri dish containing filter paper and placed in a germinator at 25°C for germination. The germinated seeds were transferred to the pots and put under controlled temperature and humidity in the greenhouse of Hirmand city, Shandel village, located 25 km from Zabul city, Sistan, and Baluchistan province. Evaluation of the effect of drought stress at four different levels of irrigation (25, 50, 75, and 100% of water requirement respectively severe stress, moderate stress, mild stress, and non-stress) and in 3 growth stages (6, 13 and 20 weeks after planting) on biochemical traits including proline content (PC), carbohydrates content (CC), total phenol content (TPC), total flavonoid content (TFC), antioxidant activity and activity of antioxidant enzymes such as catalase (CA), ascorbate peroxidase (AP), guaiacol peroxidase (GP), superoxide dismutase (SOD) and polyphenol oxidase (PO) was carried out. The experiment was done as a factorial based on a completely randomized design with three replications. Data and errors were examined for normality. After confirming the normality of the data and errors, analysis of the variance of the traits and comparing the mean of the traits (LSR) was done at the 5% level. The variance analysis of traits showed that the effect of different levels of irrigation, harvest time, and their interaction on all traits was significant. Comparison of the average interaction effect of irrigation treatment and harvest time of traits: proline content, carbohydrates content, phenol and flavonoid content, and antioxidant activity increased in all growth stages and the lowest and highest values were respectively observed in the growth stage 6 weeks after planting in 100 Percentage of water requirement and growth stage 20 weeks after harvesting in the condition of 25% water requirement. Therefore, the drought stress factor can be used to improve the effective substances of this plant. In addition, the final growth stage is the most appropriate time to harvest this plant due to the accumulation of secondary metabolites at this stage. The interaction effect of irrigation treatment and harvest time was not significant for the activity of guaiacol peroxidase enzyme, and for other antioxidant enzymes it showed that the highest activity of catalase enzyme was at the growth stage 6 weeks after planting in conditions of 25 and 50% water requirement, for ascorbate peroxidase enzyme, it belonged to the growth stage 6 weeks after planting in the condition of 100% water requirement, and for polyphenol oxidase and superoxide dismutase enzymes, it belonged to the growth stage 20 weeks after planting in the condition of 25% water requirement. These results indicate that antioxidant enzymes act differently at different growth stages and under various moisture stress conditions. The evaluation results of milk thistle in 4 irrigation regimes and three growth stages showed that most biochemical traits increased under stress conditions. This indicates that the milk thistle plant responds to drought stress through an enzymatic and non-enzymatic antioxidant defense system. Milk thistle plants had the highest total phenolic and flavonoid content at the final development stage (20 weeks after planting). Therefore, the best time to harvest is at the final stage of development, which has the most polyphenolic compounds.