The Response of Hypericum Perfpratum L. to the Application of Selenium and Nano-selenium


 Background: In terms of medicinal and therapeutic properties, H. perforatum is the important species. However, Selenium has been applied as an alleviation strategy subjected to producing essential oils and biomass.Method: For this study, a randomized complete block design with three replications was used so that each experimental unit comprised of 30 number 8 pots. The treatments included the foliar application of selenium (6, 8, 10, and 12 mg/l), nano-selenium (6, 8, 10, and 12 mg/l), and control (distilled water), applied at the rosette stage and harvesting at 50% flowering stage.Results: The results of the analysis of variance showed that the effect of selenium and nano selenium fertilizers was significant (p<0.01) on plant height, inflorescence length, number of inflorescences, inflorescence weight, shoot weight, root length, the total weight of biomass, essential oil percentage, the content of hypericin and hyperforin, the selenium accumulation in the plant, relative leaf water content, the content of chlorophylls a, b, and total, the content of phenol, the content of proline, production and accumulation of catalase, peroxidase, malondialdehyde, and DPPH enzymes. The highest inflorescence weight (21 g/plant), shoot weight (70 g/plant), and essential oil content (0.14%) were obtained from the control treatment. The highest accumulation of hypericin (3.8 mg/g dry matter) was obtained from the foliar application of 8 mg/l selenium. The maximum accumulation of hyperforin (57 mg/g dry matter) was obtained from the foliar application of 10 mg/l selenium. The highest accumulation of selenium (287.52 μg/g dry matter) was obtained in the foliar application of 12 mg/l nano-selenium.Conclusion: If the purpose of producing H. perforatum is to use the shoots and essential oils of the plant, then the use of selenium and nano-selenium is not recommended at all and should not be used. If the goal is to produce high hypericin, high hyperforin, and also the accumulation of selenium in the plant, the use of treatments of 6 and 8 mg/l of selenium and nano-selenium could be applied.


Background
Hypericum perforatum belongs to the Hypericaceae family and has more than 469 species in the world (Crockett, 2010), of which 17 (Mozaffarian, 1996) to 19 species (Zobayed et al., 2005) of this genus have been reported in Iran. St. John's wort is a perennial plant and the most important medicinal species of this genus. It is a branched plant with 40-80 cm high. The stems and branches are covered with broad leaves and soft margins, and the leaves are 1 to 3 cm long and 0.3 to 1 cm wide (Couceiro et al., 2006;Singh, 2005).
In the upper parts of H. perforatum, several yellow owers are produced, which are usually 1 to 2 cm wide. There are usually black dots on the edges of the petals. Blood red pigments are generated using crushed owers. Flowers produced in late summer have capsules that contain tiny brown seeds (Crompton et al., 1988). Although this plant is native to Europe, it is distributed in all temperate regions in Asia, Australia, and North and South America. Clinical trials have been conducted since the early 1990s on the effect of hypericin on the treatment of viral diseases, and experiments in this area show that the plant with low toxicity has signi cant effects against HIV-like viruses. So far, a wide range of biologically active compounds have been identi ed and reported in this plant, including hypericin and hyperforin.
Chemical research on the components of H. perforatum has led to the identi cation of several groups of pharmaceutically active compounds. The most important groups include naphthodianthrones (hypericin and pseudo-hypericin), phloroglucinols (hyperforin and adhyperforin), xanthones and avonoids (such as phenylpropanes, avanol glycosides and bio avonoids) as well as essential oils. Various biological activities including wound healing, anti-anxiety and seizures, antiviral, antifungal, and antioxidant properties have been attributed to the compounds in the extract and essential oil of various species of the genus Hypericum (Bertoli et al., 2011). There are various reports on the antimicrobial activity of essential oils of different species of the genus Hypericum against bacteria and fungi of human and plant pathogens.
Selenium is one of the essential elements in the human diet. Although selenium has a variety of functions, its antioxidant and anti-cancer properties are of particular importance to humans. Selenium de ciency in the human diet also causes stunted growth, impaired bone metabolism, and thyroid dysfunction (Dumont et al., 2006). Selenium concentrations are usually higher in younger leaves than in older leaves during seedling growth (Galeas et al., 2007). Because selenium is so similar to sulfur, it competes with sulfur and enters plant cells through sulfate carriers in the root plasma membrane (Pilon-Smits et al., 2002).
Nanoparticles react with plants to cause a variety of physical and physiological changes, which signi cantly depend on the properties of the nanoparticles.
The effectiveness of nanoparticles depends on their concentration and varies from plant to plant. The e ciency of nanoparticles depends on the chemical composition, size, surface area, reactivity, and concentration at which they respond positively. Nanoparticles have positive and negative effects on plant growth (Ekinci et al., 2014). Irmak (2017) conducted a study to investigate the effect of selenium application on plant growth and some quality parameters in peanut (Arachis hypogaea).
Hence, the main goal of this experiment was to evaluate the different selenium source application for with main focus morphopysiologial traits. Furthermore, we pointed to identify the appropriate concentration of Selenium that can develop essential oil and other features.

Methods
This study was conducted in 2020 in Tehran, Iran. The study aimed to survey the effect of selenium and nano-selenium on vegetative growth and phytochemical and enzymatic properties of St. John's wort in the Tehran Municipality greenhouse. This study was conducted with three replications in a randomized complete block design. Each experimental unit consisted of 30 pots. Treatments included foliar application with different levels of selenium and nano-selenium. For this purpose, selenium at concentrations of 6, 8, 10 and 12 mg/l were foliar applied at the rosette stage. In addition, nano-selenium at concentrations of 6, 8, 10, and 12 mg/l were foliar applied simultaneously with foliar application of sodium selenate. Foliar spraying of distilled water was used as a control (Fig. 1).
One-year-old seedlings of Topaz cultivar were obtained from Pakan Bazr Isfahan Company and planted in pots in March. After seedling establishment, foliar application was performed. Specimen was presented in central herbarium of Tehran University (Herbarium Code: TUH). (6398 No). Samples were authenticated by a botanist. Data collection and plant harvesting were performed at 50% owering stage. Plant height, in orescence length, number of in orescences, in orescence weight, shoot weight, root length, root weight, total biomass weight, essential oil percentage, hypericin and hyperforin content

Results
The results of the analysis of variance showed that the effect of selenium and nano selenium fertilizers on plant height, in orescence length, number of in orescences, in orescence weight, shoot weight, root length, total biomass weight, essential oil percentage, and hypericin and hyperforin content was signi cant at 1% level, and on the root weight at 5% level (Table 1). Ns, * and ** indicate non-signi cance and signi cance at the level of 5 and 1%, respectively.
The effect of selenium and nano-selenium on the amount of selenium accumulation in the plant, relative leaf water content, the content of chlorophylls a, b, and total, the phenol and proline content, and production and accumulation of catalase, peroxidase, malondialdehyde, and DPPH enzymes were statistically signi cant at the 1% level ( Table 1).
Comparison of means showed that the highest plant height was obtained from control treatment (58 cm) and foliar application of 6 mg/l selenium (56.4 cm) ( Table 2). In orescence length was the highest in control treatment and two treatment levels of 6 and 8 mg/l selenium and nano-selenium ( Table 2). The number of in orescences was the highest in the control treatment and the rst treatment level (6 mg/l) of selenium and nano-selenium (Table 2). The highest in orescence weight (21 g/plant), shoot weight (70 g/plant), root weight (27 g/plant), total biomass weight (115 g/plant), and essential oil content (0.14%) was obtained from the control treatment. The highest root weight was obtained from the control treatment (24 cm) and foliar application of 6 mg/l selenium (22 cm). The highest accumulation of hypericin (3.8 mg/g dry matter) was obtained from the foliar application of 8 mg/l selenium. The highest accumulation of hyperforin (57 mg/g dry matter) was obtained from the foliar application of 10 mg/l selenium.
It was observed that the highest accumulation of selenium (287.52 µg/g dry matter), the highest content of chlorophyll a (1.32 g), chlorophyll b (0.49 g) total chlorophyll (1.81 g), phenol (6 mg), and proline (52 µmol/g) were obtained in the foliar application of 12 mg/l nano-selenium. The highest catalase content was obtained from the foliar application of 12 mg/l nano-selenium (0.16 u/g FW/min) and foliar application of 12 mg/l selenium (0.15 u/g FW/min). Peroxidase was higher in the foliar application of 8 and 10 mg/l selenium and 10 mg/l nano-selenium than other treatments. The highest malondialdehyde leakage (695 u/g FW/min) was obtained from the 10 mg/l nano-selenium treatment. The highest levels of antioxidant enzyme (71 and 68%) were obtained from the foliar application of 8 mg/l selenium and 10 mg/l nano-selenium, respectively.
The study of simple relationships between traits showed a signi cant positive correlation between the plant height and in orescence length, number of in orescences, in orescence weight, shoot weight, root length, root weight, total biomass weight, essential oil percentage, the content of hypericin and hyperforin, relative water content, and peroxidase at the level of 1%. The plant height was also signi cantly correlated with the amount of selenium, chlorophyll a, b, and total chlorophyll, phenol, proline, catalase, malondialdehyde, and DPPH (Table 3). Similar to the plant height, the in orescence length, number of in orescences, in orescence weight, shoot weight, root length, root weight, essential oil percentage, and amount of hypericin and hyperforin had a signi cant correlation with other traits (Table 3).  Ns, * and ** indicate non-signi cance and signi cance at the level of 5 and 1%, respectively. Ns, * and ** indicate non-signi cance and signi cance at the level of 5 and 1%, respectively.

Continuous of
It was observed that the content of selenium had a signi cant negative correlation with morphological traits and yield of different organs and a signi cant positive correlation with the number of enzymes, chlorophylls, phenol, and proline. In contrast to selenium, relative water content had a signi cant positive correlation with agronomic traits and yields and a signi cant negative correlation with the number of enzymes, chlorophylls, phenol, and proline.
As can be seen in Table 3, physiological traits such as chlorophylls, enzymes, phenol, and proline had a signi cant positive correlation with each other.  (Table 1) show the selection of the hypothesis, the subject of research, and treatments, and the importance of research on H. perforatum and the introduced treatments.

Discussion
Comparison of means showed that the highest plant height was obtained from control treatment and foliar application of 6 mg/l selenium (Table 2), and the plant height decreased with increasing the concentration of treatments. The decreased height with selenium treatments indicates the intolerance of H. perforatum to selenium stress. Because the number of in orescences is affected by environmental and genetic factors and tillers, so by applying environmental treatments, the role of each of the environmental and genetic factors can be revealed. In this study, it was observed that in orescences are more affected by environmental factors. In this study, the results of the effect of selenium as an environmental factor on the number of tillers are shown in Table 2 The studied plant appears to cope with selenium and nano-selenium stress with changes in morphological traits (Thomas and Gausling, 2000), decrease in photosynthetic and transpiration organs, increase in regulatory osmolytes (Kivimäenpää et al., 2003), changes in the synthesis of materials within the plant (Flexas et al., 2009), increased water-absorbing organs (Dambolena et al., 2010) and nally allocation of photosynthetic material for active water absorption by energy (Slattery and Ort, 2015), all of which will reduce plant shoot yield as occurred for H. perforatum. The highest essential oil percentage was obtained from the control treatment, while the content of hypericin and hyperforin increased at low levels of selenium and nano-sellenium.
Basically, in plants, when they are stressed, the pentose phosphate pathway becomes more active and the production of essential oil increases. It has also been reported that plants under conditions of severe stress increase the percentage of other regulating compounds and osmolytes such as proline instead of essential oils and this is more evident in sensitive plants. The highest accumulation of hypericin and hyperforin was obtained from the foliar application of 8 mg/l and 10 mg/l selenium, respectively, showing that the use of small amounts of these two elements can be useful for producing high-quality plants.
Catalase enzyme was highest in the foliar application of 12 mg/l nano selenium and the foliar application of 12 mg/l selenium, which indicates the production of this enzyme at high levels of selenium and nano selenium. However, peroxidase production is faster than catalase in the plant, and it seems that at the same time as peroxidase production, malondialdehyde leakage reaches its maximum.
The results showed that in the foliar application of 12 mg/l nano-selenium, the maximum accumulation of selenium and the highest amount of chlorophyll a, chlorophyll b, total chlorophyll, phenol, and proline were obtained. Therefore, St. John's wort was a plant that absorbs selenium and nano-selenium, and despite the increase in chlorophyll content, a decrease in yield and an increase in antioxidant activity were observed. In general, the effect of selenium and nano-selenium particles depends on the composition, size, surface area, reactivity, and concentration in which they respond positively and have positive and negative effects on plant growth and development (Ekinci et al., 2014). In our study, the same positive and negative results were observed on different traits, and in this regard, the results are consistent with the research of Ekinci et al, (2014). The results of this study are similar to the research on Lemongrass (Melissa o cinalis L.) in terms of decreasing yield and increasing the concentration of chlorophyll a and b and the activity of catalase and malondialdehyde in the 10 mg/l treatment.
Simple relationships between traits showed a signi cant positive correlation between morphological traits and yields.

Conclusion
Therefore, if the purpose of producing H. perforatum is to use the shoots and essential oils of the plant, then the use of selenium and nano-selenium is not recommended at all and should not be used. If the goal is to produce high hypericin, high hyperforin, and also the accumulation of selenium in the plant, the use of treatments of 6 and 8 mg/l of selenium and nano-selenium could be used. A signi cant negative correlation between selenium content with morphological traits and yield of different organs and a positive correlation with the content of enzymes, chlorophylls, phenol, and proline shows that selenium causes stress in the plant and therefore plant growth is reduced and instead increases the production of antioxidant enzymes and osmolytes. There is a signi cant negative correlation between selenium content with morphological traits and function of different organs and a positive correlation with the number of enzymes, chlorophylls, phenol and proline indicates that selenium causes stress in the plant and therefore plant growth is reduced and instead production increases antioxidant enzymes and osmolytes. Availability of data and materials: Not available