*3.3. Priming Effect of SPM on Electrolyte Leakage, Total Soluble Sugar and Total Soluble Protein under Cr Stress*

In control conditions (seed priming with water and SPM without Cr toxicity), no noticeable changes in the values of electrolyte leakage (EL), total soluble sugar (TSS) and total soluble protein (TSP) were observed in the leaves of both rice cultivars (Figure 2a–c). However, Cr stress alone caused a prominent increase in the values of EL (78.4% in CY927, and 43.7% in YLY689) (Figure 2a) but decreased the TSS and TSP levels (49.2/36.5% in CY927, and 37.4/31.2% in YLY689, respectively) (Figure 2b,c), when compared to the control without Cr stress. The increasing or decreasing trends of EL, TSS and TSP values were more pronounced in CY927 (in the case of EL) than YLY689 (in the case of TSS and TSP), although results represented that seed priming with SPM reduced the EL (32.9% and 45.7%), and increased the TSS and TSP by 24.7/19.3% in CY927 and 32.2/21.6% in YLY689 cultivar, respectively, when compared to their relative controls under Cr stress (Figure 2a–c). These outcomes indicated that SPM notably minimized the EL but improved the TSS and TSP levels compared to seed priming with water, especially in YLY689 under Cr stress. These outcomes suggested that SPM notably reversed the Cr-mediated induction in EL and reduction in TSS, as well as TSP in rice seedlings.

**Figure 1.** Effects of seed primed SPM on photosynthetic pigments (**a**) chlorophyll–a, (**b**) chlorophyll–b, (**c**) total chlorophyll–(a + b), and (**d**) carotenoids, (**e**,**f**) *Fv*/*Fm* levels in the leaves of two rice cultivars CY927 and YLY689, respectively and (**g**,**h**) *Fm* levels in the leaves of two rice cultivars CY927 and YLY689, correspondingly under chromium (Cr) stress. Values are mean ± SE of three independent replicates and different letters (a–f) above bars show a significant difference between treatments by LSD test at *p* < 0.05.

#### *3.4. Priming Effect of SPM on Hydrogen Peroxide (H2O2), Superoxide (O2* •−*) and Malondialdehyde (MDA) under Cr Stress*

Under Cr alone treatments, the accumulation of H2O2 (91.2/72.5% and 67.1/55.8%), O2 •− (76.9%/67.1% and 51.8/47.3%), and MDA (77.6/74.2% and 59.2/46.9%) were significantly induced in shoots/roots of CY927 and YLY689, correspondingly, as compared to control plants. Inclusively, a high accumulation of H2O2, O2 •− and MDA was noticed in shoots rather than roots of both cultivars (Figure 2d–i). The accretion of H2O2, O2 •− and MDA contents were more prominent in CY927 than in YLY689, though seed priming with SPM markedly decreased the accumulation of H2O2 (38.8/27.3% and 33.4/24.9%), O2 •− (35.2/26.3% and 24.9/18.3%) and MDA (47.8/37.1% and 38.2/23.7%) in shoots/roots of both varieties (CY927 and YLY689), correspondingly, under Cr stress (Figure 2d–i). In control treatments, no significant difference was noticed among the H2O2, O2 •− and MDA content values in the roots and shoots of both rice cultivars.

**Figure 2.** Effects of seed primed SPM on (**a**) electrolyte leakage (EL), (**b**) total soluble proteins, (**c**) total soluble sugar (TSP), (**d**) MDA contents in shoots, (**e**) MDA contents in roots, (**f**) H2O2, contents in shoots (**g**) H2O2, contents in roots (**h**) O2 •− contents in the shoots and (**i**) O2 •− contents in the roots of two rice varieties (CY927 and YLY689) against chromium (Cr) stress. Values are mean ± SE of three independent replicates and different letters (a–f) above bars show a significant difference between treatments by LSD test at *p* < 0.05.

To verify the accumulation of H2O2 and O2 •− inside the shoots and roots of both cultivars of rice, leaves were stained with DAB and NBT in response to seed priming with water, SPM and Cr treatments (Figure 3a–h). Compared to untreated control, leaves treated with Cr only revealed dark brown as well as dark blue staining, correspondingly, for H2O2 and O2 •−. CY927 exhibited more dark staining colors than YLY689 indicating that CY927 accumulates more H2O2 or O2 •− than YLY689. In contrast, control treatments (seed primed with water and SPM without Cr addition) displayed a slight staining intensity of DAB and NBT in both cultivars (Figure 3a–h). These differences revealed that seed priming with SPM sustained the plasma membrane integrity and reduced the oxidative damages against Cr-induced overproduction of ROS in both rice cultivars (particularly in YLY689) in comparison to seed priming with water against Cr toxicity conditions.

**Figure 3.** Effects of seed primed SPM on histochemical staining to identify the accumulation of (**a**) hydrogen peroxide (H2O2) in the shoots of CY927, and (**b**) hydrogen peroxide (H2O2) in the shoots of YLY689 by 3,3-diaminobenzidine (DAB), (**c**) superoxide (O2 •−) in the shoots of CY927, and (**d**) superoxide (O2 •−) in the shoots of YLY689 by nitro blue tetrazolium (NBT), (**e**) hydrogen peroxide (H2O2) in the roots of CY927, and (**f**) hydrogen peroxide (H2O2) in the roots of YLY689 by 3,3-diaminobenzidine (DAB), (**g**) superoxide (O2 •−) in the roots of CY927, (**h**) superoxide (O2 •−) in the roots of YLY689 by nitro blue tetrazolium (NBT).
