Morpho-physiological and phytochemical responses of basil (Ocimum basilicum L.) to toxic heavy metal cadmium

Cadmium (Cd) is a particularly noteworthy metal that may change the secondary metabolism efficacy of pharmaceutical valuable plants. This study aimed to explore the influence of Cd (0, 25, 75, 100, and 150 μM) on the morpho-physiological traits, mineral contents, essential oil composition, and morphology of secretory glands in basil (Ocimum basilicum L.). The exposure to Cd reduced significantly shoot and root dry weight, shoot and root lengths, total chlorophyll, number, and length of secretory gland compared to control plants, while increased peroxidase activity, proline content, and number of stomata. Essential oil compositions were varied in the Cd-treated plants in a dose-dependent manner. The main compounds of the essential oil were Estragole followed by Linalool and Geranial which the highest percentages were observed in the plants treated with Cd 150 μM. The Cd-treated plants exhibited reductions in calcium (Ca) and iron (Fe) concentrations in both leaves and roots. Cd contents in roots and shoots progressively enhanced with increasing Cd concentrations In general, basil showed good tolerance to Cd stress, so that 50% reduction in shoot dry weight occurred in above 100 μm treatments, which can be related to physiological reactions such as increased antioxidant activity, proline content, number of leaves stomata, and changes in other morphophysiological factors.


Introduction
Taking agriculture into account, environmental pollutants, in particular, heavy metals (the nonbiodegradable elements) have become a significant concern (Chmielowska- Bąk et al., 2018;Iranbakhsh et al., 2018). These pollutant agents might adversely influence various living organisms, like plants and humans. Environmental contamination with pollutants like cadmium (Cd) is a common issue that may restrict the crop productivity. Cd is a major toxic heavy metal with an unknown biological role in human life (Fattahi et al., 2019). Cd is a high-mobility element, and exposure to Cd can be, therefore, highly toxic (Chmielowska-Bąk et al., 2018). In this concern, various attempts have been made to mitigate Cd-induced phytotoxicity . It is well established that Cd is commonly associated with increases in the overproduction of reactive oxygen species (ROS) and subsequent induction of oxidative stress in cells (Chmielowska-Bąk et al., 2018). In adaptive response to heavy metals, reception, and signal transduction are followed by changes in transcriptions of genes , organ development (Melato et al., 2012), and metabolism (Babajani et al., 2019b).
Common basil (Ocimum basilicum L.) as a member of the Lamiaceae family is an aromatic multifunctional vegetable with economical great essential oils (terpenoid derivatives) (Alemu et al., 2018).
Essential oils are secondary metabolites with isoprenoid structure. The accumulation of essential oils in plants is usually limited to specialized secretory structures which are present in several different types, namely glandular trichomes. As shown in Figure 1, leaves secretory glands to consist of several cells providing different functions. The essential oils secreted by secretory cells are passed through a space situated at the apex of the subcuticular trichome and get accumulated; however, the basal cells provide attachment to the skin structure (Hazzoumi et al., 2017). Basil essential oil compounds are exploited in the food and medicine industries owing to their multiple functions, especially antimicrobial, antioxidant, flavor, perfume, and preservative traits (De Araujo Couto et al., 2019). Besides, these natural terpenoid-derivatives displayed outstanding characteristics to control parasites, fungi, bacteria, viruses, and insects (Predoi et al., 2018). Furthermore, diverse isoprenoids exhibit considerable pharmaceutical functions to cure several diseases, including malaria, Alzheimer's, cardiovascular disorders, and cancer (Lu et al., 2016). Figure 1. Glandular structure, drawn from a photograph by Gershenzon et al. (1989) The content and chemical composition of the basil essential oils has been the subject of many studies. The yield from different plant parts varies between 0.2-1.9% with the main components being linalool, methyl chavicol, eugenol, and methyl cinnamate, as well as 1,8-cineole, methyl eugenol, geraniol, geranial, neral and αbergamotene (Ilic et al., 2019). Fattahi et al. (2019) reported that sweet basil cultivation in the Cd contaminated soils could cause an undesirable effect on the morphological traits, but might have a positive influence on the essential oil yield, composition, and phytoremediation of the soil.
In plants, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are active isoprene units by which a multitude of terpenoids during the secondary metabolism are synthesized ( Figure 2). Productions of these C5 precursors (isoprenes; IPP and DMAPP) are mediated through two independent routes, including the mevalonic acid (MVA) and the methylerythritol phosphate (MEP) (Lu et al., 2016).
Prenyl transferases are enzymes that contributed to the condensation process of C5 isoprene units by which structure of terpenoid derivatives, including monoterpenoids, diterpenoids, sesquiterpenoids, and triterpenoids are formed (Lu et al., 2016).
Most research has focused on the mechanisms of Cd-induced phytotoxicity. However, there is little documented convincing evidence toward the possible Cd effect on secondary metabolism, and in particular, the quality of essential oils which have an industrial interest. Furthermore, tissue differentiation and organ development epigenetically may be influenced by environmental factors, like irrigation, nutrition, pollutant, etc. (Seddoghinia et al., 2019;Moghanloo et al., 2019a). The negative impact of contaminated soil with heavy metals on plant and human health is an important global concern. In this concern, Cd may cause variations in morphology and distribution of secretory structures in leaves. Hence, the aim of this experiment was an examination of the possible influence of different Cd concentrations ranging from 25-150 µM on the growth, distribution of leaves secretory glands, essential oil composition, and nutritional status in basil. population were purchased from Pakan Seed Co., Isfahan, Iran. The seeds were sterilized using sodium hypochlorite and sown in a pot containing soil with traits described in Table 1. Seedlings were kept in a greenhouse under controlled temperature (26 °C ± 4 °C), humidity (50 ± 10%), photoperiod (16/8 h light/dark), and light intensity (160 μmol/m-2/s-1). Following this, each pot was irrigated every 2 days (for ten times) with half strength of Hoagland's nutrient solution containing various levels of cadmium chloride (CdCl2.5H2O) (25, 75, 100, and 150 µM). Experiments were designed in a completely randomized design (CRD) with three replicates. Four-week-old plants were harvested to measure all morphological, anatomical, and physiological parameters. Total chlorophyll content In order to measure the total chlorophyll content, the samples were immediately frozen in liquid nitrogen and kept in a freezer at -80 °C before conducting the biochemical analyses. Then, the photosynthetic pigments were measured by using Lichtenthaler (1987) method. Based on this method, 0.25 g of fresh tissue was extracted by using 5 ml 80% acetone. Then, the extract was centrifuged at 11000 rpm for 10 min. In addition, the optical density (O.D.) of the extract was measured at the wavelengths of 646.8 and 663.2 nanometer.

Peroxidase activity
To measure the enzyme activity of the total seedling, 0.1 g of fresh tissue was used. To extract protein, 0.2 g of fresh tissue plant was pulverized in a mortar using liquid nitrogen and then one mL of buffer Tris-HCl (0.05 M, pH = 7.5) was added. The obtained mixture centrifuged for 21 min at 13,000 rpm, at 4 °C and the supernatant was used for enzyme activity measurements (Aghighi Shahverdi et al., 2017). The peroxidase activity was determined in a reaction of the mixture, which consisted of a suitable amount of 28 mM guaiacol, 5 mM H2O2, 25 mM Na-phosphate buffer (pH 6.8) and enzyme (Ghanati et al., 2002).
Proline content Proline was determined according to the method described by Bates et al. (1973). Approximately 0.5 g of fresh seedling tissue was homogenized in 10 ml of 3% aqueous sulfosalicylic acid. Then, this aqueous solution was filtered through Whatman's paper No. 2 and finally, 2 ml of filtrated solution was mixed with 2 ml acidninhydrin and 2 ml of glacial acetic acid in a test tube. The mixture was placed in a water bath for 1 h at 100 °C. The reaction mixture was extracted with 4 ml toluene, cooled to room temperature, and the absorbance was measured at 520 nm with a spectrometer.

Essential oil analysis
Essential oil samples were analysed using GC-MS (GC-2010 SHIMADZU), according to the method described in detail for the type, column, etc. by Fattahi et al. (2016). Basil leaves were collected and hydrodistilled using a Clevenger apparatus. Gas chromatography/mass spectrometry (GCMS) analysis was equipped with a split-splitless injector (split ratio; 30:1), scan time 1s, ionization energy 70 eV, and mass range of 40-300 amu. a column (60 m × 0.25 mm i.d., film thickness 0.25 m); oven temperature was 60-230 °C at a rate of 7 °C/min, transfer line temperature 260 °C. The carrier gas was helium, with a linear velocity of 31.5 cm/s. The oils diluted in dichloromethane (2 µl of oil in the 2 ml solvent), the next 2µl oil of each treatment was injected to GC/MS manually. The retention indices were calculated, for all volatile constituents using a homologous series of n-alkanes C8-C22 on the HB-5 column. The essential oil constituents were determined by comparing their GC retention indices, mass spectra with data published in the literature of Adams. Compounds were further identified using their mass spectra data compared National Institute of Standards and Technology mass spectra library data provided by the software of the GC-MS system. Essential oil components are shown as a relative percentage of the total oil.

Morphology and distribution of leaf secretory glands and stomata
Leaves secretory glands consist of several cells providing different functions. The Cd-mediated changes in morphology and distribution of leaf secretory glands were monitored by Scanning Electron Microscope (SEM) (Hazzoumi et al., 2017). To measure the number of leaf stomata, the Radoglou and Jarvis (1990) method was used. For each leaf sample, 8 observations of a light microscope (Leica Galen III) in magnification 200 were prepared. The number of leaf stomata by GSA Image Analyzer V3.8.6 Software was determined.

Statistical analysis
Distribution normality of achieved data was done according to the Kolmogorov-Smirnov and Shapiro-Wilk test. Then the studied traits were statistically analysed by the Statistical Analysis System software (SAS Institute, Cary, NC, USA, and Version 9.2). The differences among means were separated using LSD test (least significant difference) at 0.05 statistical probability level.

Results
As shown in Table 2, the shoot and root lengths reduced significantly with the increasing Cd concentrations. This decrease in Cd150 treatment was 23.9 and 59.7% compared to the control treatment, respectively. However, there was no significant difference in root lengths between Cd100 and Cd150 treatments. Plant shoot dry weight decreased significantly in basil plants exposed to different concentrations of Cd. Plants subjected to 25, 75, and 100 µM Cd showed a reduction of 14, 28.5, and 39% of plant dry weight, respectively, relative to the control plants. The strongest decrease in plant shoot dry weight (57%) was observed at the highest Cd concentration. The exposure of basil seedlings to 25 to 150 µM Cd reduced significantly the root dry weight compared to the control plants (Table 2). The effect of Cd stress was significant on physiological traits, number of stomata, and secretory gland characteristics (Table 3). The Cd 150 treatment was caused decreases in total chlorophyll (51.0%), number of secretory glands (63.0%), and secretory gland length (16.9%) while increased peroxidase activity (71.4%), proline content (62.8%), and number of stomata (28.2%), in compared to the control treatment (Table 3). As shown in Figures 3 and 4, both distribution and morphology of secretory glands in leaves were influenced by Cd treatments in a dose-dependent manner.
Ca, Fe, and Cd contents in basil leaves and roots are presented in Table 4. Changes in Ca concentrations were more pronounced in leaves when compared to roots. The Cd-treated plants exhibited reduced Ca concentrations in both leaves and roots. In leaves and roots, Fe contents were influenced showing a significant decline in all Cd-treated plants. Cd contents in roots and shoots progressively were enhanced with increasing Cd concentrations. In general, with increasing Cd concentrations, the content of Fe and Ca absorption decreased in both root and stem organs, while the Cd content increased in both organs.
Results of the simple correlation presented in Table 5. According to the results, shoot dry weight showed a significantly positive correlation with shoot length, root length, root dry weight, total chlorophyll, number of secretory glands, secretory gland length, root, and shoot Ca contents, shoot and root Fe contents, while negatively correlated peroxidase activity, proline content, number of stomata, shoot and root Cd contents.    The results of GC-MS analyses of the essential oil are shown in Table 5. Geranial concentration increased by Cd 25, 75, and 150 treatments, however, Cd100 showed a decrease over the control plant. The δ-Amorphene concentration recorded a progressive increase in Cd 25, 75, and 100 treatments compared to the control. On the contrary, Cd 150 declined δ-Amorphene content. The application of increasing Cd concentrations (25, 75, 100, and 150 µM) increased cubenol concentration. Linalool did not vary at 25 and 75 µM Cd treatments, while a significant decrease occurred at 100 µM. Our results showed that Cd enhanced Caryophyllene oxide production in basil and this effect was dose-dependent. Level of 25, 75, and 150 µM Cd did not induce changes in δ-Cadinene compared to the control, while 100 µM Cd increased the content of δ-Cadinene. The application of 25, 75, and 100 µM Cd-induced an increase of the α-Bisabolene and Βcaryophyllene percentage. At 25 and 75 µM Cd, no changes in the percentages of Calamenene and Neral occurred.

Discussion
Cd stress reduced growth parameters and biomass accumulation. The results of the present study showed that a 50% reduction in the shoot dry weight (as the economic part of this plant) occurs in more than 100 µM Cd, and this indicates the optimal tolerance of this plant to the Cd stress. This can be due to physiological mechanisms such as the increased activity of antioxidant enzymes (peroxidase), proline content, number of leaves stomata. The overall results of this study showed that the presence of Cd in the culture medium affected the yield and growth characteristics of basil and reduced growth and disruption of various physiological processes in plant structure. It has been reported that the effect of Cd in peppermint reduced leaf area and dry weight of the aerial parts in Cd in comparison with control in peppermint (Amirmoradi et al., 2012) and basil (Fattahi et al., 2019).
With a similar trend, Cd treatments, especially at the high doses considerably decreased Ca and Fe concentrations in both leaves and roots. These results are in line with several other reports in different plant species, including wheat (Abbas et al., 2018;Rizwan et al., 2019), rice , tomato (Ahmad et al., 2018b), and okra (Rasheed et al., 2018). Cd-induced disruption of membrane permeability, oxidative stress, photosynthesis performance, and nutrition may result in a decrease in plant growth. Hong et al. (2015) indicated that heavy metals prevent not only plant growth, but also adversely affect nutrient content. Heavy metals, including Cd, may inhibit or reduce the uptake of elements, including Cu, Fe, and Zn thereby blocking some enzyme activation and negatively affecting photosynthesis. Hence, the inhibitory effect of heavy metals on water relations, nutrient uptake, and photosynthesis might lead to a decline in plant growth (Kilic et al., 2017). Cd irreversibly prevents proton pumps involving in cell elongation and division owing to bonding with nucleophilic groups and consequent membrane permeability disruption. Moreover, Cd negatively affects photosynthetic pigments and the ultrastructure of the chloroplast, leading to decreased carbon assimilation (Ahmad et al., 2018a).
Plants have antioxidant mechanisms to reduce the damage caused by ROS including non-enzymatic components such as ascorbate, glutathione, carotenoids, proline, and anthocyanin, and enzymes such as CAT, SOD, POD, etc. (Suzuki and Katano, 2018). Proline is an amino acid stored in the cytoplasm and is likely to protect macromolecules' structure inside the cell during stress conditions. Proline concentration in stress conditions may increase up to 100% under normal conditions. This osmoprotectant plays an essential role in regulating cell osmotic pressure under stresses such as drought, salinity, and toxicity (Slama et al., 2015). Based on our results, the proline content and peroxidase activity were increased as a consequence of Cd stress.
Our results confirmed that the chemical composition of essential oils was altered in response to Cd treatments. Induction in secondary metabolism following exposure to Cd may be attributed to Cd-mediated changes in ROS levels and their subsequent signalling. It is well established that exposure to Cd is commonly associated with increases in the overproduction of reactive oxygen species (ROS) and subsequent induction of oxidative stress in cells (Chmielowska-Bak et al., 2018). In addition to the possible ROS-mediated oxidation of biomolecules, these bioactive compounds can trigger specific signalling pathways and alter gene expression and cellular metabolism Babajani et al., 2019a;Babajani et al., 2019b). In response to heavy metal stress, ROS over accumulations associate with redox signalling, thereby changing developmental processes in plants (Berni et al., 2018). In line with our results, heavy metals altered the chemical composition of secondary metabolites in Melissa officinalis (Babajani et al., 2019b), Hypericumper forum (Murch et al., 2003), and Cymbopogon citratus (Lermen et al., 2015). Changes in the chemical composition of secondary metabolites may affect the medicinal efficacy of these natural resources. Many studies reported linalool as the main compound of investigated basil essential oils Elgndi et al., 2017). Sharafati-Chaleshtori et al. (2015) reported methyl chavicol as the main compound of basil essential oils while eugenol was detected as the compound with the highest abundance by Piras et al. (2018). In the present study, all of the compounds listed above had significant increases in the high level of Cd concentration.
Moreover, exposure to Cd changed both the morphology and distribution of the secretory gland. It has been well documented that tissue differentiation may vary in response to various environmental cues (Moghanloo et al., 2019b;Seddiginia et al., 2019). Mizushima et al. (2019) indicated that Avicennia schaueriana can exclude toxic Cd in the form of Cd/Ca crystal through secretion of leaf salt glands, representing a fast mechanism for the Cd release. Choi et al. (2001) also showed that tobacco can eliminate Cd by excreting the Ca/Cd crystals through the trichome cells. Therefore, the Cd-associated changes in differentiation of secretory tissues may be considered as a potential defensive mechanism. Taken collectively, soil contamination with Cd may not only reduce growth but also may influence secondary metabolism and tissue differentiation.

Conclusions
Here, we determined the effect of Cd exposure on the morpho-physiological characteristics and the concentration of Ca, Fe, and Cd in various parts of basil. Application of 150 µM Cd reduced the morphophysiological traits such as shoot and root length, shoot and root dry weight, total chlorophyll, number of secretory glands, secretory gland length, and concentration of Ca and Fe in the shoot and root organs, but increased peroxidase activity, proline content, number of stomata, and root and shoot Cd contents. In general, Cd stress increased the average essential oil composition of this plant compared to Cd-free treatment. Higher levels of Cd (100 and 150 µM) also showed the highest percentage of essential oils in the plant.