Morphophysiological and Biochemical Responses of Crocus sativus ‎During the Interaction of Melatonin and Drought Stress

Melatonin increases stress tolerance in plants. Saffron's red stigmas are the main Iran's industrial and export products. The aim of this study was to investigate the effect of melatonin on some morphophysiological and biochemical characteristics of saffron under drought-stress conditions. Drought stress was applied by polyethylene glycol 6000 at 10% and 20% levels. Root treatment of melatonin was applied at a concentration of 100 μM. Drought stress, particularly 20% stress, led to a decrease in growth parameters. Under 20% stress, the amount of chlorophyll a, chlorophyll b, carotenoids, and photosynthesis rate decreased by 22.32%, 35.84%, 51.35%, and 10.05%, respectively, while the ion leakage (2.70%), proline content (15.00%), and flavonoid (1.94%) increased. Exogenous application of melatonin has a positive effect on increasing plant resistance to drought stress by affecting physiological indicators such as photosynthetic parameters and the antioxidant system.

Drought is one of the limiting and effective abiotic factors in agricultural productivity in the world. Under drought stress conditions, plant growth factors decrease (Zhang et al., 2014; Brunner et al., 2015). Drought stress leads to the accumulation of reactive oxygen species in cells. Excessive accumulation of ROS causes lipid peroxidation and ion leakage. By inactivating enzymes, proteins, and nucleic acids, ROS reduces photosynthesis and the yield of crops (Altaf et al., 2022). Plants use different strategies to respond to stress. An increase in abscisic acid levels, compatible osmolyte content, protective enzyme activities, and antioxidant levels with inhibiting energy consumption pathways are among these strategies (Li et al., 2018). Natural or synthetic growth regulators such as melatonin can affect plant physiological processes at low concentrations. Melatonin regulates the biological responses of plants by modulating various physiological, biochemical, and molecular processes and finally increases the resistance of plants against drought conditions. Saffron with the common name Saffron and the scientific name Crocus sativus is from the Iridaceae family. As the most expensive agricultural and medicinal product in the world, saffron has a special place among Iran's industrial and export products (Farooq et al., 2009). Since most of the regions of Iran are facing the problem of water shortage, studying the responses of plants to water stress is of particular importance. The aim of this study was to investigate the potential improving effects of melatonin on the antioxidant and photosynthetic responses of Crocus sativus L. under drought-stress conditions. 

After disinfection, the corms were washed and transferred to pots containing perlite. Irrigation and addition of Hoagland's nutrient solution were done at one-week intervals. In order to apply drought stress, polyethylene glycol 6000 was used at two levels of 10% and 20%. Root treatment of melatonin with a concentration of 100 μM was done after applying drought stress. The fresh and dry weight of the samples, the relative water content, and the amount of photosynthetic pigments were measured according to the methods of Moradi Rikabad et al. (2019), Martı̀nez et al. (2004), and Arnon (1949), respectively. The maximum photosystem quantum efficiency (Fv/Fm) II was measured using a fluorometer apparatus (Pocket PEA) and the amount of net photosynthesis (Pn) was measured using a portable gas exchange device model CI-340. In order to measure proline content, ninhydrin was used as a reagent, and proline was used as a standard. The sugar in the samples was measured by the phenol-sulfuric acid method. The leakage of electrolytes was measured by the method of Lutts et al. (1996). The amount of H2O2 was calculated using the extinction coefficient of 0.28 mmol/cm. The measurement of phenolic and flavonoid compounds was done by colorimetric method using Folin-Ciocaltue reagent and aluminum chloride, respectively. The free radicals inhibition was measured by the DPPH method. This research was conducted as a factorial study in the form of a completely randomized design in three replications. To analyze the data, Minitab 16.0 was used, and the significance levels of differences between samples were determined using Tukey's test at the P ≤ 0.05 statistical level. Microsoft Office Excel 2013 was used to draw the graphs.

The results of the study showed that drought stress decreases the height, fresh and dry weight, and relative water content of the plant. Exogenous melatonin improves plant growth factors in these conditions. Melatonin probably stimulates plant growth factors by affecting plant metabolites and increasing the biosynthesis of phytohormones and facilitating the absorption of nutrients (Arnao, 2014; Naghizadeh et al., 2019). According to the results, drought stress causes a decrease in the content of photosynthetic pigments, Pn, and Fv/Fm. In fact, drought stress disrupts the balance between the absorption capacity and the use of light energy, causing disruption in photosynthesis, and damage to the photosynthetic apparatus (Pinheiro & Chaves, 2011). Exogenous melatonin treatment protects chlorophyll against abiotic stress by reducing lipid peroxidation and regulating the downstream genes of chlorophyll degradation (Yu et al., 2018). Exogenous treatment of melatonin by modulating the stomatal pore can also improve plant growth and increase Pn under drought stress (Liu et al., 2020). In this study, drought stress led to an increase in proline and soluble sugars, although these changes were not significant.
The Exogenous application of melatonin in these conditions decreased the content of these osmolytes. Under stressful conditions, the accumulation of compatible osmolytes improves turgor pressure, thereby preventing cell dehydration (Liang et al., 2018). The Exogenous application of melatonin may maintain the water balance in the plant through other ways and thus regulate the cell turgor pressure and reduce the content of compatible osmolytes (Ibrahim et al., 2020, Altaf et al., 2022). The results showed that drought stress leads to an increase in hydrogen peroxide content and ion leakage, although these changes were not significant, exogenous application of melatonin caused a decrease in hydrogen peroxide content and ion leakage while the content of phenol and flavonoid were increased. Drought stress often causes the accumulation of reactive oxygen species, which results in cell membrane damage. Under stressful conditions, melatonin can increase the activity of the H+-ATPase pump and contribute to the stability of the plasma membrane (Wang et al., 2019).

The results of this study showed that melatonin increases the resistance of Crocus sativus L. against drought stress by regulating the physiological and biochemical responses. In today's conditions, where water scarcity is one of the most important challenges in world agriculture, the use of new growth regulators, including melatonin, can be effective in increasing the tolerance of plants to drought stress and improving the growth and quality of agricultural products.