*2.3. Rohitukine Treatment for A. thaliana*

Five-week-old plants of *A. thaliana* with uniform growth and maturity were exposed to 0.25 mM, 0.5 mM and 1.0 mM concentrations of pure rohitukine. Ten plants were treated with each concentration, and plants sprayed with autoclaved double-distilled water without rohitukine were used as controls. The plants were sprayed thrice with the appropriate concentrations of rohitukine dissolved in 50 mL autoclaved double-distilled water (1 mL to each plant) at intervals of 24 h, and 1 h post-treatment the samples were collected for analysis. Rohitukine is polar in nature, with a water solubility of >10 mg/mL and lipophilicity (log D) of <1.0 [10].It was found to have pH-dependent solubility, with its highest solubility in simulated gastric fluid (SGF) [45]. The reported value of acidity constant (pKa) is 5.83 [10]. We prepared the spray reagent at around pH 7.0 and it is therefore expected to be present at unionized form in solution, leading to higher absorption/permeation.


**Table 1.** Composition of MS nutrient media used for the growth of *A. thaliana* plants.

#### *2.4. Determination of Rohitukine Content in A. thaliana*

The content of rohitukine in treated samples was determined by thoroughly washing an equal quantity (200 mg) of rosette leaves, followed by homogenization in HPLC grade methanol and sonication, after which the homogenate was centrifuged at 12,000 rpm for 10 min at room temperature (RT). The supernatant was filtered through a syringe filter of 0.22 μm pore size and analysed following the protocol of Kumar et al. (2016) [43], using a HPLC system (Shimadzu, UFLC) consisting of a quaternary pump with a vacuum degasser, a thermos tatted column compartment, an autosampler and a PDA detector. A reverse-phase column (Lichrosphere RP C18e, 5 μm, 250 mm× 4 mm) was used and the column temperature was maintained at 40 ◦C. The HPLC mobile phase consisted of two solutions. Solution A was composed of water with 0.1% formic acid. The solution was filtered through a 0.45 μm membrane filter and degassed in a so nicator for 3 min. Solution B was pure HPLC-grade acetonitrile. The mobile phase was run using gradient elution: 0.01 min, 10% B; in the next 20 min, 50% B; in the next 5 min, 70% B; in the next 5 min, 90% B, and maintained at 80% B for 5 min; in the next 5 min, 10% B; followed by an equilibration period of 5 min. The flow rate was 0.8 mL/min, and the injection volume was 20 μL. The eluents were detected and analysed at 254 nm. A quantity of 1mg/mL of pure rohitukine was taken as standard.

#### *2.5. Measurement of Leaf Area and Plant Weight*

Leaf area was measured using the easy leaf area tool described by Easlon et al. (2014) [46]. Briefly, *A. thaliana* rosette leaves treated with rohitukine were photographed by placing them on plain paper along with a red-coloured piece of paper of 4 cm2, and measurements were made using easy leaf area software by following the instructions provided. The fresh weight of the aerial parts (rosette leaves) of plants was determined immediately after harvesting. For dry weight, the samples were completely dried in an oven until weight

was constant, then weighed to obtain the dry weight using a fine weighing balance (Mettler Toledo, Columbus, OH, USA).

#### *2.6. Photosystem II Activity*

Chlorophyll fluorescence parameters were assessed for the rohitukine-treated plants of *A. thaliana* using a Junior-PAM chlorophyll fluorometer (Heinz Walz, Effeltrich, Germany), following the manufacturer's instructions. The parameters assessed were: actual photosystem II (PSII) efficiency (Φ PSII), intrinsic PSII efficiency (Fv/Fm), maximum PSII efficiency (Fv/Fm), photochemical quenching (qP), non-photochemical quenching (NPQ) and electron transport rate (ETR). ETR was calculated usingthe formula Φ PSII × photosynthetic photon flux density × 0.5 × 0.84, as given in [47].

#### *2.7. Photosynthetic Pigment Quantification*

Total chlorophyll content was determined in *A. thaliana* plants exposed to 0.25 mM, 0.5 mM and 1.0 mM rohitukine by adopting the method of Arnon (1949) [48], with minor modifications. Briefly, 200 mg fresh leaf samples were homogenized in 0.2 mL 80% acetone, followed by centrifugation at 12,000 rpm for 5 min. The supernatants were collected and their absorbances were recorded at 663 and 645 nm for chlorophyll content using a spectrophotometer.

#### *2.8. Histochemical Detection of ROS*

The accumulation of H2O2 and O2 − was analysed by the histochemical staining method, as described by Shi et al. (2010) [49], using 3,3-diaminobenzidine (DAB) (Sigma-Aldrich, St. Louis, MO, USA, cat. no. D5637) and nitro blue tetrazolium (NBT) (Hi Media, cat. no. RM578), respectively. For H2O2 detection, a solution of 1mg/mL DAB was prepared in 10 mM phosphate buffer, and the pH was adjusted to 3.8 with1N HCL. *A. thaliana* leaves of similar maturity levels were immersed in DAB solution at RT in the dark until brown spots were visible. After six hours of staining, the samples were incubated in a mixture of ethanol, acetic acid and glycerol (3:1:1) at 80 ◦C for 15 min to bleach out chlorophyll for proper visualization. ForO2 − localization, the samples were immersed in a 1 mg/mL solution of NBT prepared in 10 mM phosphate buffer (pH 7.8) until blue-coloured spots appeared. The immersed samples were then boiled in ethanol for better visualization, and photographs were taken by placing the stained leaves in a clean place with a white background using a digital camera.

#### *2.9. The Activity of Antioxidant Enzymes*

Fresh leaf tissue of rohitukine-treated and untreated (control) samples of *A. thaliana* were homogenized in potassium–phosphate extraction buffer (100 mM, pH 7.0), using a precooled pestle and mortar. The extraction buffer was composed of 0.05% (*v*/*v*) Triton X-100 and 1% (*w*/*v*) polyvinylpyrrolidone (PVP). The activity of ascorbate peroxidase (APX; EC, 1.11.1.11) was calculated following the protocol described by Asgher et al. (2014) [50]. Briefly, APX activity was determined by the decrease in absorbance of ascorbate at 290 nm due to its enzymatic breakdown. A total of 1 mL mixture of 50 mM phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM EDTA, 0.1 mM H2O2 and enzyme extract was prepared. The calculation of APX activity was carried out using the extinction coefficient 2.8 mM−<sup>1</sup> cm<sup>−</sup>1, and one unit of enzyme was required to decompose 1 μmol of substrate per min at 25 ◦C.

#### *2.10. RNA Extraction, cDNA Synthesis and qRT-PCR*

Rohitukine-treated leaves of *A. thaliana* were used as source material for RNA isolation, using TRizol reagent (Ambion, Life Technologies, Carlsbad, CA, USA), according to the manufacturer's protocol. RNA quality and concentration were assessed on a 2% agarose gel and nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) by measuring the absorbance ratio at 260/280 nm. The isolated RNA samples were subjected to DNase (Ambion TURBO DNA-free, Life Technologies, Carlsbad, CA, USA) treatment to remove traces of genomic DNA. DNase-treated RNA samples were reverse-transcribed using a cDNA synthesis kit (Promega, Madison, WI, USA), following the manufacturer's instructions, with oligo (dT) primers and 1μg of DNase-treated RNA used as a template. Primer pairs were designed from coding sequences (CDSs) of selected antioxidant system genes of *A. thaliana* (Table 2). The qRT-PCR reactions were set in the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), based on SYBR green chemistry. The PCR reaction mixtures (10 μL) comprised SYBR Green Master mix at a volume of 5.0 μL, 1.0 μM of each primer (Integrated DNA Technologies, Coralville, IA, USA), appropriately diluted cDNA as template and MQ water was used to make up the final volume of 10 μL. Thermoprofiles of the reactions for qRT-PCR were included by preincubation at 95 ◦C for 10 min, followed by 45 cycles of 3-step amplification with melt (95 ◦C for 10 s, 60 ◦C for 10 s and 72 ◦C for 15 s) and melt. The normalization of the reaction was achieved using the primers of the actin gene as a control. The analysis of samples was carried out in triplicate, and the specificity of each primer pair was authenticated by a dissociation curve (a single peak was observed for each primer pair). The generated (threshold cycle) CT values were then transferred to Microsoft Excel and the 2−ΔΔCT method of relative quantification was used to determine the quantitative variation between the samples examined [51].

**Table 2.** Table showing the details of primers used for gene expression analysis.

