Cd causes inhibitory effects on crop growth and such effects are generally concentration-dependent and genotype-specific. In this study, Cd treatment severely inhibited the seed germination of both rice cultivars, which was consistent with previous study . The ZnO NPs treatment showed no notable effect on the germination of rice (Fig. 1A-B). In a previous study, the effect of nanomaterials like polystyrene nanoplastics was reported, and they were described to have a prominent effect on seed germination and early growth of wheat .
Additionally, application of ZnO NPs mitigated the Cd toxicity on the growth of rice seedlings, promoting an increase in seedling weight, particularly in the shoot as well as the total fresh weight, while also promoting an increase in the root-shoot ratio (Fig. 2). However, the effect of all ZnO NPs treatment on the growth of rice seedling remained statistically similar (p > 0.05) (Fig. 2). Previous studies have reported the negative effect of Cd on the seed germination, root length, shoot, and biomass of rice seedlings [13, 21]. However, the effect of ZnO NPs on the growth of rice remains controversial. For instance, the positive effect of 50 ppm ZnO NPs treatment on the growth of rice was identified , whereby ZnO NPs treatment with 500 ppm and 1000 ppm concentrations had negative effects on the early growth of rice. In contrast, 25, 50 and 100 mg L− 1 ZnO NPs treatments had a negative effect on the growth of rice seedlings.32 So, exogenous application ZnO NPs in a suitable range/concentration would be able to promote the growth of rice, whereas high concentrations could have inhibitory effects.
Starch is an important storage polysaccharide for energy involved in the growth of rice seedlings. A previous study showed that α-amylases could facilitate both seed germination and seedling growth by mobilizing nutrients in the endosperm, however, starch would be immobilized under the Cd toxicity, which resulted in growth inhibition . The exposure to Cd or ZnO NPs-based treatment had no significant effect on the α, β and total amylase activity (Fig. 3). The ZnO NPs treatment increased the α-amylase and total amylase activity under Cd stress (Fig. 3), which may be beneficial for the metabolism of stored substances in rice grains, and may improve the early growth of rice.
Moreover, seed priming with ZnO NPs regulated the antioxidant enzyme activity i.e., SOD, POD, CAT, as well as MT and MDA contents in both rice cultivars. Normally, SOD reduces O2− to H2O2 and O2, whereas CAT scavenges the H2O2 generated during the photorespiration and β-oxidation of fatty acids . The POD is located in cytosol, vacuole, cell wall, as well as in extracellular space, and uses guaiacol as an electron donor, while utilizing the H2O2 in the oxidation of various inorganic and organic substrates. Cd toxicity enhances the production levels of ROS, which increases lipid peroxidation, resulting in an increased generation of MDA as a byproduct [46, 47]. Besides, in order to mitigate the negative effect of ROS, the antioxidant enzyme activities increased at lower Cd concentrations but decreased at higher Cd levels [47–49]. In this study, Cd treatment reduced the SOD activity, but increased the POD and CAT activity, along with the MDA content in the root and shoot of the seedling (Figs. 4 and 5A-B). A previous study showed that ZnO NPs treatment reduced the ROS generation, and induced higher activities of antioxidant enzymes, i.e., SOD, CAT and POD . In this study, ZnO NPs treatment only increased the POD activity in shoot for Xiangyaxiangzhan (Fig. 4). Under Cd treatment, the ZnO NPs treatment had notable effect on the SOD, POD and CAT activity (Fig. 4). Other than the antioxidant enzymes, MT is one of the most potent bioactive substances that scavenges ROS , and is utilized by plants to decrease the heavy metal concentration . MT content in root was increased by the Cd treatment, while no significant increase was noted in shoot (Fig. 5C-D). A higher MT content in shoot of rice seedlings under ZnO NPs and Cd treatments was also noted (Fig. 5C-D). Overall, under Cd stress, seed priming with ZnO NPs treatment activated stress-resistance, while promoting the MT formation, and reducing the MDA accumulation in the plant.
Furthermore, ZnO NPs treatment of 50 mg L− 1 showed substantial growth improvement of the rice seedlings under 100 mg L− 1 Cd stress. Therefore, the metabolites in the shoot of rice seedlings under the four treatments (A: ZnO NPs 0 + Cd, B: ZnO NPs 0 + Cd 100, C: ZnO NPs 50 + Cd 0, and D: ZnO NPs 50 + Cd 100) were assessed (Additional file 3: Table S2). Overall, there were 26 metabolites which had VIP values > 1, which could be considered as the main compounds for distinguishing the treatments in both cultivars. For Xiangyaxiangzhan, there were 26 metabolites that had VIP values > 1, while there were 40 metabolites that had VIP values > 1 in Yuxiangyouzhan (Fig. 6). There were 20 important metabolic pathways identified in this study which were affected by the treatment (Fig. 7).
The regulations in glutathione metabolism in response to the toxicity of Cd described in this study are consistent with previously reported . Moreover, amino acid-, purine-, carbon-, and glycerolipid- metabolic pathways were also affected by the Cd stress, which resulted in reduction of plant growth and/or photosynthetic capacity, and promoted the defense mechanisms to minimize cell damage . The comparative analysis of the metabolites between the treatments with varied Cd and ZnO NPs doses allowed identification of metabolites regulating ‘zeatin biosynthesis’, ‘purine metabolism’, ‘pyrimidine metabolism’, ‘glutathione metabolism’, ‘cysteine and methionine metabolism’, ‘arginine and proline metabolism’, ‘biosynthesis of unsaturated fatty acids’, ‘caffeine metabolism’, and ‘flavonoid biosynthesis’/ ‘stilbenoid, diarylheptanoid and gingerol biosynthesis’ (Fig. S1 and S2). Among the treatments, ZnO NPs 0 + Cd 100 and ZnO NPs 50 + Cd 100 elicited pronounced change in metabolites, while the ZnO NPs 50 + Cd 0 showed marginal changes in metabolites. Change in a few other metabolites, such as (5-l-glutamyl)-l-amino acid was detected in both the rice cultivars (Additional file 5: Table S4). The changes of these metabolites showed a consistent pattern with concomitant changes in growth and physiological parameters in response to the ZnO NPs under Cd stress.
Moreover, according to the analysis of the differential metabolites, high levels of L-Ascorbic acid, γ-glu-cys, and oxidized glutathione were detected in A vs B and A vs D for both cultivars, while no significant change was observed in A vs C (Additional file 1: Fig. S1 E, Additional file 4: Table S3). Ascorbic acid (ASA) and glutathione (GSH) cycle are important pathways for a plant to cleanse the ROS . The ascorbate peroxidase (APX) enzymes neutralize the H2O2 into H2O by utilizing ascorbate as an electron donor, which is subsequently oxidized to form monohydroascorbic acid (MDHA). The AsA-GSH cycle system may use AsA/DHA, GSH/GSSG, and NAD(P)H/NAD(P) to maintain an appropriate redox environment in plants [54, 55]. Besides, γ-glu-cys is utilized to synthesize GSH via the glutathione synthase pathway. The accumulation of γ-glu-cys is an important way for plant to protect itself under Cd stress . Overall, seed priming with ZnO NPs treatment had a positive effect on the antioxidant enzymes of rice seedling under Cd stress. Notably, xanthine was detected in high concentrations in shoot of the rice seedling in A vs C, and A vs D for both rice cultivars. Thus, the ZnO NPs treatment may affect the xanthine-related response pathways which are actively involved in the synthesis of DNA or RNA.