*3.3. Effect of Rohitukine on Plant Morphology*

The *A. thaliana* plants treated with rohitukine showed concentration-dependent growth inhibition as compared to the control. A significant decrease in leaf area of 17.7%, 25.1%, and 60.3% with the0.25 mM, 0.5 mM and 1.0 mM rohitukine concentrations, respectively, was observed as compared to the untreated group. The fresh weight of plants treated with 0.25 mM, 0.5 mM and 1.0 mM of rohitukine decreased by 11.6%, 17.4% and 38.7%, respectively. Similarly, dry weight was found to be reduced by 12.5% 19.5% and 40.2% with 0.25 mM, 0.5 mM and 1.0 mM rohitukine, respectively (Figure 3). The maximum growth inhibitory effect of rohitukine was observed in *A. thaliana* plants treated with a 1.0 mM concentration.

**Figure 3.** Morphology of *A. thaliana* plants treated with T0 (Control), T1 (0.25 mM), T2 (0.5 mM) and T3 (1.0 mM) rohitukine concentrations. (**a**) Five-week-old *A. thaliana* plants with and without rohitukine treatment. (**b**) Effects of 0.25 mM, 0.5mM and 1mM Rohitukine on leaf area of *A. thaliana* plants. (**c**) Average fresh weight of plants. (**d**) Effect of 0.25 mM, 0.5mM and 1mM Rohitukine on the dry weight of *A. thaliana.* The results are the means of more than three replicates expressed as means ± SD values. Statistical significance was determined by Student's *t*-test. Asterisks \* and \*\* denote the significance level of values at *p*-values < 0.5 and 0.05, respectively.

#### *3.4. Influence of Rohitukine on Chlorophyll Content and PSII Activity*

Total chlorophyll content and PSII activity were studied in *A. thaliana* exposed to different concentrations of pure rohitukine by comparing it with untreated control plants. Plants treated with rohitukine showed a dose-dependent decrease in chlorophyll content. With 0.25 mM, 0.5 mM and 1.0 mM rohitukine concentrations, total chlorophyll content decreased by 1.8, 1.7 and 1.6 mg/g fresh weight (FW), respectively, as compared to the untreated control plants having 2.5 mg/gFW (Figure 4g). Actual PSII efficiency (Φ PSII), maximum PSII efficiency (Fv/Fm), intrinsic PSII efficiency, photochemical quenching (qP) and electron transport rate (ETR) decreased by 6.9%, 4.81%, 9.21% and 5.19%, respectively, under 0.25 mM rohitukine. On the other hand, non-photochemical quenching (NPQ) increased by 26.4% compared to the control. Application of 0.5 mM rohitukine decreased Φ PSII, Fv/Fm, Intrinsic PSII efficiency, qP and ETR by 9.7%, 6.02%, 10.52% and 7.79%, respectively, and non-photochemical quenching increased by 32% when compared to the control. Plants that received 1.0 mM rohitukine exhibited maximally decreased Φ PSII, Fv/Fm, Intrinsic PSII efficiency, qP and ETR, by 25%, 21.68%, 18.42% and 18.18%, respectively, and a 54% increase in NPQ as compared to the control (Figure 4).

**Figure 4.** Effects of 0.25 mM, 0.5 mM and 1.0 mM rohitukine on (**a**) actual PSII efficiency, (**b**) maximum PSII efficiency, (**c**) intrinsic PSII efficiency, (**d**) photochemical quenching, (**e**) non-photo chemical quenching, (**f**) electron transport rate and (**g**) total chlorophyll content in five-week-old *A. thaliana* plants treated with rohitukine. Data are presented for the treatments as means ± SDs (*n* = 3). Statistical significance was determined using Dunnett's multiple comparisons test. Asterisks \*, \*\* and \*\*\* denote significance level at *p*-values < 0.5, 0.05 and 0.01, respectively.

#### *3.5. Accumulation of O2* − *and H2O2 in A. thaliana Plants in Response to Rohitukine*

Increased levels of O2 − were noticed in *A. thaliana* plants as dark blue-coloured spots scattered on the leaves treated with rohitukine as compared to the untreated control. Similarly, the *A. thaliana* plants treated with pure rohitukine showed a significant increase in brown-coloured spots when immersed in DAB staining dye. These scattered brown spots were indications of increased levels of H2O2 in rohitukine-treated leaves as compared to untreated samples (Figure 5). Asin animal cells, rohitukine induces ROS in plant tissues, with maximum ROS observed for the1.0 mM treatment.
