Soil applied silicon and manganese combined with foliar application of 5-aminolevulinic acid mediate photosynthetic recovery in Cd-stressed Salvia miltiorrhiza by regulating Cd-transporter genes

Abstract

Salvia miltiorrhiza is an important medicinal plant that experiences significant growth and biomass losses when cultivated on cadmium (Cd) contaminated soils. High Cd accumulation in plant tissues also increases the risk of metal entry into the food chain. In this study, we proposed that Cd accumulation in S. miltiorrhiza can be restricted through plant growth regulators and nutrient management. Therefore, S. miltiorrhiza seedlings were transplanted into mixed nutrient soil for two weeks, then treated with 30 mg kg^-1 CdCl₂, 200 mg kg^-1 Na₂SiO₃·9H₂O, and 100 mg kg^-1 MnSO₄, and simultaneously sprayed with 10 mg L^-1 ALA on the leaves one week later. This study showed that elevated Cd accumulation significantly reduced plant growth and biomass. This growth inhibition damaged photosynthetic machinery and impaired carbon assimilation. In contrast, 5-aminolevulinic acid (ALA) significantly promoted the biomass of S. miltiorrhiza, and the dry weight of plants treated with ALA combined with manganese (Mn)/silicon (Si) increased by 42% and 55% as compared with Cd+Mn and Cd+Si treatments. Exogenously applied ALA and Si/Mn significantly activated antioxidant enzymes and promoted the growth recovery of S. miltiorrhiza. Further, exogenous ALA also reduced the Cd concentration in S. miltiorrhiza, especially when combined with Si. Compared with the Cd+Si treatment, the Cd+Si+ALA treatment reduced the Cd concentration in roots and leaves by 59% and 60%, respectively. Gene expression analysis suggested that ALA and Si significantly up-regulated genes associated with Cd transport. Other genes related to heavy metal tolerance mechanisms are also regulated to cope with heavy metal stress. These results indicated that the combined action of ALA and Si/Mn could reduce Cd-toxicity by increasing chlorophyll content and changing oxidative stress and can also affect Cd accumulation by regulating gene expression.

Keywords: gene regulation; heavy metals; manganese; oxidative stress; plant growth regulator; silicon.

Figure 1

Effects of application of 5-aminolevulinic acid (ALA), silicon (Si), manganese (Mn), ALA combined with Mn/Si on S. miltiorrhiza plants growth under cadmium (Cd) stress. Concentrations of Cd, ALA, Si and Mn were 30 mg kg^-1 CdCl₂, 10 mg L^-1 ALA, 200 mg kg^-1 Na₂SiO₃·9H₂O and 100 mg kg^-1 MnSO₄, respectively.

Figure 2

Effects of application of ALA and Mn/Si on levels of reactive oxygen species and lipid peroxidation in the roots and leaves of Cd stress plants. Hydrogen peroxide (H₂O₂) in the roots (A) and leaves (B); superoxide radical $\left({\text{O}}_2^{-}\right)$ in the roots (C) and leaves (D); hydroxyl ion (^-OH) content in the roots (E) and leaves (F); malondialdehyde (MDA) in the roots (G) and leaves (H). Concentrations of Cd, ALA, Si and Mn were 30 mg kg^-1 CdCl₂, 10 mg L^-1 ALA, 200 mg kg^-1 Na₂SiO₃·9H₂O and 100 mg kg^-1 MnSO₄, respectively. Data are the means of three replicates (mean ± SE). Small letters indicate significant differences at P< 0.05 by Duncan’s multiple range tests.

Figure 3

Effects of application of ALA and Mn/Si on antioxidant enzyme activities in the roots and leaves of Cd stress plants. Superoxide dismutase (SOD) in the roots (A) and leaves (B); peroxidase (POD) in the roots (C) and leaves (D); catalase (CAT) in the roots (E) and leaves (F); ascorbic acid peroxidase (APX) in the roots (G) and leaves (H); glutathione reductase (GR) in the roots (I) and leaves (J). Concentrations of Cd, ALA, Si and Mn were 30 mg kg^-1 CdCl₂, 10 mg L^-1 ALA, 200 mg kg^-1 Na₂SiO₃·9H₂O and 100 mg kg^-1 MnSO₄, respectively. Data are the means of three replicates (mean ± SE). Small letters indicate significant differences at P< 0.05 by Duncan’s multiple range tests.

Figure 4

Effects of application of ALA and Mn/Si on Cd accumulation under Cd stress plants. Cd accumulation in the roots (A), Cd accumulation in the leaves (B), Cd bioaccumulation quantity (BCQ) in the roots (C), Cd bioaccumulation quantity in the leaves (D). Concentrations of Cd, ALA, Si and Mn were 30 mg kg^-1 CdCl₂, 10 mg L^-1 ALA, 200 mg kg^-1 Na₂SiO₃·9H₂O and 100 mg kg^-1 MnSO₄, respectively. Data are the means of three replicates (mean ± SE). Small letters indicate significant differences at P< 0.05 by Duncan’s multiple range tests.

Figure 5

Expression maps of nine resistance-related genes of S. miltiorrhiza. Concentrations of Cd, ALA, Si and Mn were 30 mg kg^-1 CdCl₂, 10 mg L^-1 ALA, 200 mg kg^-1 Na₂SiO₃·9H₂O and 100 mg kg^-1 MnSO₄, respectively. Data are the means of three replicates (mean ± SE). The expression level of the control group was normalized as “1”. Drawing heat map using expression data processed by log10.

Figure 6

Effects of ALA and Mn/Si application on the expressions of tolerant related genes in S. miltiorrhiza roots under Cd stress. Concentrations of Cd, ALA, Si and Mn were 30 mg kg^-1 CdCl₂, 10 mg L^-1 ALA, 200 mg kg^-1 Na₂SiO₃·9H₂O and 100 mg kg^-1 MnSO₄, respectively. Data are the means of three replicates (mean ± SE). Small letters indicate significant differences at P< 0.05 by Duncan’s multiple range tests.

Figure 7

Effects of ALA and Mn/Si application on the expressions of tolerant related genes in S. miltiorrhiza leaves under Cd stress. Concentrations of Cd, ALA, Si and Mn were 30 mg kg^-1 CdCl₂, 10 mg L^-1 ALA, 200 mg kg^-1 Na₂SiO₃·9H₂O and 100 mg kg^-1 MnSO₄, respectively. Data are the means of three replicates (mean ± SE). Small letters indicate significant differences at P< 0.05 by Duncan’s multiple range tests.

Figure 8

Effects of ALA and Mn/Si application on cryptotanshinone, tanshinone I, tanshinone IIA and salvianolic acid B under Cd stress plants. Concentrations of Cd, ALA, Si and Mn were 30 mg kg^-1 CdCl₂, 10 mg L^-1 ALA, 200 mg kg^-1 Na₂SiO₃·9H₂O and 100 mg kg^-1 MnSO₄, respectively. Data are the means of three replicates (mean ± SE). Small letters indicate significant differences at P< 0.05 by Duncan’s multiple range tests.

Figure 9

Scanning electron microscope (SEM) results of MnSO₄ image at 30 μm (A), MnSO₄ image at 10 μm (B), MnSO₄ image at 2 μm (C), Na₂SiO₃·9H₂O image at 200 μm (D), Na₂SiO₃·9H₂O image at 10 μm (E), Na₂SiO₃·9H₂O image at 2 μm (F).