*3.6. Effect of Rohitukine on Antioxidant Enzyme Activity*

The activity of APX, SOD and POD increased by 2.9%, 2.8% and 6.6%, respectively, with the 0.25 mM rohitukine treatment, whereas with the0.5 mM treatment, the activity of APX, SOD and POD increased by 4.47%, 4.34%, and 17.7%, respectively. The application of the 1.0 mM rohitukine concentration maximally increased APX activity by 10.44%, SOD by 13.04% and POD by 31.1% as compared to the untreated control (Figure 6).

**Figure 5.** The effects of rohitukine concentrations on T0 (control), T1 (0.25 mM), T2 (0.5 mM) and T3 (1.0 mM) as detected through histochemical detection of ROS in *A. thaliana* leaves. Superoxide ions were detected with NBT staining dye; the H2O2 visualization in leaves was performed via DAB staining.

**Figure 6.** Effects of 0.25 mM, 0.5 mM and 1.0 mM rohitukine on (**a**) APX, (**b**) SOD and (**c**) POD of five-week-old *A. thaliana* plants treated with rohitukine. Data are presented for the treatments as means ± SDs (*n* = 3). Statistical significance was determined by Dunnett's multiple comparisons test. Asterisks \* and \*\* denote significance level at *p*-values < 0.5 and 0.05, respectively.

#### *3.7. Effect of Rohitukine on the Expression of Key Genes Involved in the Antioxidant System*

The expression patterns of key genes involved in the antioxidant system, such as manganese Mn-SOD, Cu/Zn-SOD, APX and POD, were analysed at transcript level in rohitukine-treated *A. thaliana* samples. The significantly increased expression of these genes was noticed in treated samples as compared to untreated controls. The Mn-SOD gene was found to have a two-fold increased expression in the 0.25 mM and 0.5 mM rohitukine treatments, while in the 1.0 mM treatment we noticed around a three-fold expression of Mn-SOD when compared to controls. Similarly, APX gene transcripts were found to have six-fold upregulated expression in the 0.5 mM and 1.0 mM treatments with rohitukine. In the treatment with the 0.25 mM concentration, the APX was found to be upregulated by 3.5-fold as compared to the control. In the treatment with the 1.0 mM rohitukine concentration, significantly higher transcripts of Cu/Zn-SOD were noticed in *A. thaliana* as compared to the control, whereas with the 0.25 and 0.5 mM rohitukine concentrations, the expression of Cu/Zn-SOD was found to be higher as compared to the control. The expression of the POD gene was also upregulated by rohitukine. The 0.5 mM and 1.0 mM concentrations of rohitukine increased the expression of POD by 2.5 and 4.5-fold, respectively (Figure 7). In all the treatments, overall expression profiles of Mn-SOD, APX, Cu/Zn-SOD and POD were increased when compared to controls. However, plants exposed to 1.0 mM of rohitukine showed the highest transcript levels for all the genes examined.

**Figure 7.** qRT-PCR-based expression analysis of key genes of the antioxidant system of *A. thaliana*. The bar diagrams represent the fold change expression of each gene upon 0.25 mM, 0.5 mM and 1 mM rohitukine exposure as compared to untreated controls. The results are presented as the means of three replicates and as means ± SDs. Statistical significance was determined by Student's *t*-test. Asterisks \* and \*\* denote the significance of fold changes at *p*-values < 0.05 and 0.005, respectively, as compared to untreated controls.

#### *3.8. Metabolite Profiling*

With GC–MS analysis of rohitukine-treated *A. thaliana* leaf samples after derivatization and comparison of identified spectra with the NIST17 library, a total of 75, 73, 70 and 71 metabolites were identified with their known structures in the control, 0.25 mM, 0.5 mM and 1.0 mM rohitukine-treated samples, respectively. The metabolites identified were fatty acids, sugars, amino acids, organic acids, polyamines, carbohydrates, etc. To obtain the robust metabolome datasets, only those metabolites which were found to be present in at least three replicates were considered, and their presence in all four samples was ensured before processing. A total number of 37 metabolites were found to be common to all four samples (0.0 mM, 0.25 mM, 0.5 mM and 1.0 mM) and in each replicate. To understand the differences between samples and the similarity between replicates, and to determine the variables that contributed most to these differences, principal component analysis (PCA) was carried out for 37 metabolites, where PCA1 showed 41.8% and PCA2 showed 24.9% of the variation. The accumulation patterns of metabolites in the control as well as in all three treatments are shown by a heatmap generated from 37 metabolites present in all the samples (Figure 8). In all four major clusters, distinct patterns of altered levels of metabolite were reported. Therefore, the patterns of metabolite abundance and clustering indicate the metabolic changes caused by the exposure to rohitukine in *A. thaliana* leaves.

**Figure 8.** Heatmap illustration of quantities of commonly found metabolites in *A. thaliana* after 0.25 mM, 0.5 mM and 1.0 mM rohitukine treatments as compared to controls. The colour intensities of each box represent the level of each metabolite in each rohitukine-treated group.

Among sugars, the most frequent members found in all the four samples were: erythrose, fructose, galactose, glucose, maltose and sucrose. Dose-dependent decreased levels of fructose, glucose, maltose, sucrose and galactose were noticed in rohitukine-treated samples as compared to controls. Amino acids, L-alanine, aspartic acid, L-threonine and L-tyrosine were found with significantly higher concentrations in rohitukine-treated *A. thaliana* plants when compared to controls (Figure 8). Some other metabolites which were found to be significantly higher after rohitukine exposure were 3-alpha-Mannobiose, ethanolamine and silanamine. Meanwhile, the accumulation of several metabolites was found to be significantly lower after rohitukine exposure, including glycerol, Myo-inositol, acetamide, diethylamine, pentasiloxane, etc. Moreover, we also noticed changes in the levels of several organic acids in rohitukine-treated samples; the upregulated organic acids

included: lactic acid, trihydroxy butyric acid and 4-amino butanoic acid. Conversely, several organic acid metabolites, such as palmitic acid, boric acid, stearic acid and oxalic acid, were found to have significantly lower concentrations in the 0.5 mM and 1.0 mM rohitukine treatments.

#### **4. Discussion**

Although the biological activities of rohitukine in mammalian as well as in yeast strain cells have been deeply studied, the significance of this molecule in plant systems has not yet been elucidated. It is for the first time that we have tried to understand the physiological and biochemical impacts of rohitukine inside the *A. thaliana* model system. However, the rohitukine biosynthesis pathway in parent plants has not been elucidated yet. As far as the accumulation of rohitukine in source plant *D. gotadhora* is concerned, it was reported to accumulate in leaves, bark, fruits, seeds and twigs, but the highest concentration of rohitukine was reported in seeds (2.42%), followed by leaves (1.06%) [15,54]. Later, Kumar et al. (2016) [10] introduced the chromatography-free protocol of rohitukine isolation, in which they extracted 98% pure compound with a 1% (dry weight) yield. In the present study, we isolated 1.6 g of rohitukine from 200 g dry leaves of *D. gotadhora* with 98% purity (Figure 1). The pure compound of rohitukine was extracted from *D. gotadhora* (source plant) and applied to *A. thaliana*, where rohitukine interfered with plant growth and development. It is possible that rohitukine may leach out from the leaves of the parent plant during rain and it could also be found in soil samples as an allelochemical, which may also be due to the decomposition of plant tissues in soil. The leaching of toxic alkaloids from plant tissues into soil and drainage water has also been evidenced by previous studies [55,56].

For screening of the best inhibitory/modulatory concentration of rohitukine, we applied a range of rohitukine concentrations (0.01 mM to 10 mM) on *A. thaliana* seedlings. Among the concentrations screened so far, 1.0 mM rohitukine maximally inhibited the growth of *A. thaliana*, while 0.25 mM and 0.5 mM concentrations moderately affected growth as scored visually. Therefore, we applied 0.25 mM, 0.5 mM and 1.0 mM concentrations of rohitukine to analyse the dose-dependent effects of the molecule on *A. thaliana*. In previous studies, the dose-dependent growth inhibitory effects of extracts from an important medicinal plant *Hyptissuaveolens* were examined in several plant species [57].The stability and uptake of the compound were also assessed through HPLC, and rohitukine was found to be stable inside the plant tissues and uptake was also confirmed (Figure 3) [10,43]. The question related to the stability of rohitukine at various physiological pH levels is very important for its phytotoxic efficiency. In already published reports, the stability of rohitukine has been assessed by incubating the pure compound of rohitukine in buffers of different pH (1.2, 4.0, 6.8 and 7.4), bio-relevant fluids, such as SGF (pH 1.2) and simulated intestinal fluid (SIF) pH 6.8, and also in rat plasma. Rohitukine was found to be stable in all the tested conditions [10,43].

It has been reported that rohitukine is highly stable in diverse biological fluids. Moreover, it is found to be in the category of high-permeability molecules (log papp > −5) based on Parallel Artificial Membrane Permeability Assay (PAMPA) data, which confirm passive transcellular permeation [52]. Computational analysis of rohitukine suggests that it is a substrate of P-gp, which is the key member of the ABC transporter system [45]. In addition, stress situations are reported to regulate transporter expression in the plant system [58]. The above-mentioned evidence in the literature indicates the possible uptake of rohitukine in biological systems. However, further investigations have to be performed to explore the exact mechanism of rohitukine uptake in *A. thaliana* [59]. Additionally, the plasma protein binding efficiency of rohitukine and its semisynthetic derivatives have also been reported [60]. However, in plants, the exact mechanism of membrane transport of rohitukine is unclear. An attempt has been made, using isolated epidermal cells, to understand the mechanism of alkaloid transport through plasma membranes, suggesting the involvement of transporter proteins [61].

The negative effects of plant secondary metabolites, such as L-mimosine, syringaldehyde, juglone, and vanillin, have already been assessed for *A. thaliana* and other crop species [62–64]. In the present study, rohitukine exposure significantly decreased total leaf area and plant biomass. Moreover, a moderate decrease in photosynthesis was observed with 0.25 and 0.5 mM rohitukine concentrations, while photosynthesis decreased maximally in plants that received 1.0 mM of rohitukine when compared to the controls. Similarly, a gradual decrease in total chlorophyll content was observed with increased rohitukine concentration. Similar results have been shown by Hussain and Reigosa (2021) [65], who investigated the influence of two plant secondary metabolites, ferulic acid and phydroxybenzoic acid, on the photosynthesis of *Rumex acetosa*, where both the molecules inhibited photosynthetic parameters, such as Fv/Fm, Φ PSII, qP and NPQ. Rutin is another secondary metabolite that is reported to inhibit Fv/Fm and the concentrations of chlorophyll pigments in *A. thaliana* [66]. At increased rohitukine concentrations, we noticed a decrease in ETR, Φ PSII, Fv/Fm, qP and intrinsic PSII efficiency in *A. thaliana*. Consequently, increased NPQ was observed upon rohitukine exposure.

Generally, ROS are produced inside living cells when they encounter any external stress and activate the antioxidant defence system to overcome the such caused by oxidative stress [67]. In plants, ROS are present in •O2 <sup>−</sup> ionic states, such as hydroxyl radicals (•OH), and molecular states, including H2O2 and singlet oxygen (•O2) [68,69]. •O2 − is reported to increase during external stress and is the precursor of various ROS. The excessive generation of •O2 − causes an increment in ROS that leads to cell death [70,71]. Rohitukine exposure affected various physiological, biochemical and molecular mechanisms via the excessive production of ROS in *A. thaliana*. In rohitukine-treated *A. thaliana* plants, we detected the accumulation of •O2 − and H2O2 as blue- and dark brown-coloured spots, respectively, through the histochemical staining of leaves. The already published literature suggests that rohitukine induces ROS in yeast strains after 24 h of treatment [16]. Moreover, in cell lines, rohitukine and its semisynthetic derivatives have been reported to induce ROS-mediated apoptosis [21,72,73]. Similarly, two other alkaloids, Graveoline and vitrine, isolated from *Ruta graveolens* and *Evodilitoris*, with immunomodulatory, anti-inflammatory and anticancerous activities are reported to generate ROS in the root coleoptile of wheat [74,75]. We also noted the rohitukine-mediated induced expression of APX, SOD and POD, which are involved in the antioxidant defence system in plants. These genes are exclusively associated with ROS metabolism to combat the stress response. Therefore, it is likely that rohitukine may trigger cell death in plants via ROS generation and affect hormonal transport. Under all defined concentrations of rohitukine, there was a significant increment in APX, SOD and POD enzyme contents in *A. thaliana*. Khan et al. (2011) [76] investigated the SOD-, CAT- and POD-mediated growth inhibitory effects of aqueous and ethanol extracts of *Peganum multisectum* on ryegrass.

The reduction in maximum quantum yield of PSII shows that excitation energy trapping of PSII reaction centers was reduced. A decrease in photosynthesis was suggested to be due to stomatal closure [65]. Reduction in CO2 passage due to stomatal closure is responsible for the accumulation of ROS, the degradation of xanthophyll pigments and lipids and protein oxidation [77]. The slight decrease in PSII activity observed was due to more ROS accumulation, which increases oxygen production at 1 mM rohitukine. Moreover, the slightly higher ETRs recorded for treatments with 0.25 mM and 0.5 mM of rohitukine compared to the 1 mM treatment were due to less ROS accumulation, as revealed by DAB and NBT staining, which limits ROS production. Our results also show that rohitukine can block the electron acceptor to inhibit photosystem II. These results support the hypothesis that there was a reduction in photosystem II photochemistry and photosynthetic electron transport, which is responsible for ROS accumulation in *A. thaliana*. Similarly, secondary metabolites isolated from several endophytes inhibit PSII electron transport on the water-splitting enzyme and on the acceptor side between P680 and QA. The results of this study were confirmed by chlorophyll-a fluorescence measurements [78]. The reduction in chlorophyll fluorescence under metal stress shows HM antenna pigment

disruption due to the hindrance of electron transport flow from PSII to PSI [79–81]. To address the phytotoxic effects of secondary metabolites, many studies have been published which show that secondary metabolites have impacts mainly through damage to photosynthetic machinery and frequent decomposition of photosynthetic pigments. Consequently, decrease in photosynthetic pigments leads to blockage of energy/electron transfer and inhibition of ATP synthesis [28].

The changes caused by any elicitor at the morphological and transcriptomic levels should be reflected in the final end products of gene and protein expression. The metabolome offers better visualization of the changes in the levels of many different metabolites. In recent years, metabolomics has gained considerable attention as a tool for acquiring better insight into the biological processes of organisms [82–85]. In an attempt to analyse the changes in metabolite levels caused by rohitukine in *A. thaliana*, GC–MSbased metabolomics was performed, in which amino acids, carbohydrates, other organic acids, etc., were identified. Among amino acids, L-alanine, aspartic acid, L-threonine and L-tyrosine were detected at higher concentrations in 0.5 mM and 1.0 mM rohitukine-treated samples. Generally, aromatic amino acids, such as tyrosine, play important roles in the synthesis of a wide range of secondary metabolites when plants encounter any external stress. L-tyrosine and other aromatic amino acids act as precursors for the synthesis of phenylpropanoids, a major group of plant secondary metabolites whose function is to protect the plant from abiotic stresses [86–88]. Many essential amino acids, such as valine, methionine, alanine, and leucine, as well as non-essential amino acids, such as histidine, proline cysteine, etc., have been reported to increase under abiotic and biotic stresses [89–91]. The metabolic analysis also identified dose-dependent decreases in concentrations of some sugars, including fructose, glucose, maltose, sucrose and galactose, in rohitukine-treated *A. thaliana* samples as compared to controls. The maximum decreased level of sugars was noticed with the1.0 mM rohitukine treatment. Disruption to photosynthesis and chlorophyll bleaching by rohitukine exposure in *A. thaliana* plants could be the reason for lesser concentrations of sugars in treated samples. There are several studies that have shown the negative impacts of extracts and pure compounds isolated from different plant species on recipient plants in the form of decreased levels of carbohydrates [92–94]. The concentration of myo-inositol was found to be slightly decreased upon rohitukine exposure. It has been reported that Myo-inositol participates in cellular functions and metabolism in plants [95]. It also mediates ROS-induced cell death in the presence of salicylic acid and ethylene towards stress tolerance [96]. Along with Myo-inositol, several other metabolites, such as acetamide, diethylamine, penta siloxane and glycerol, were found to have decreased levels. Indole-3-acetamide triggers stress responses in *A. thaliana* and participates in the crosstalk of auxin and abscisic acid [97].
