The E3 Ubiquitin Ligase Gene Sl1 Is Critical for Cadmium Tolerance in Solanum lycopersicum L

Abstract

Heavy metal cadmium (Cd) at high concentrations severely disturbs plant growth and development. The E3 ubiquitin ligase involved in protein degradation is critical for plant tolerance to abiotic stress, but the role of E3 ubiquitin ligases in Cd tolerance is largely unknown in tomato. Here, we characterized an E3 ubiquitin ligase gene Sl1, which was highly expressed in roots under Cd stress in our previous study. The subcellular localization of Sl1 revealed that it was located in plasma membranes. In vitro ubiquitination assays confirmed that Sl1 had E3 ubiquitin ligase activity. Knockout of the Sl1 gene by CRISPR/Cas9 genome editing technology reduced while its overexpression increased Cd tolerance as reflected by the changes in the actual quantum efficiency of PSII photochemistry (Φ_PSII) and hydrogen peroxide (H₂O₂) accumulation. Cd-induced increased activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) were compromised in sl1 mutants but were enhanced in Sl1 overexpressing lines. Furthermore, the content of Cd in both shoots and roots increased in sl1 mutants while reduced in Sl1 overexpressing plants. Gene expression assays revealed that Sl1 regulated the transcript levels of heavy metal transport-related genes to inhibit Cd accumulation. These findings demonstrate that Sl1 plays a critical role in regulating Cd tolerance by relieving oxidative stress and resisting heavy metal transportation in tomato. The study provides a new understanding of the mechanism of plant tolerance to heavy metal stress.

Keywords: antioxidant enzymes; heavy metal stress; protein degradation; tomato; ubiquitination.

Figure 1

The relative expression level of Sl1 in tomato plants. (A) The relative expression level of Sl1 in different tissues of tomato. (B) Time course of the relative expression level of Sl1 in tomato root with and without Cd stress. The data presented here are the average of three biological replicates (±SD). Different letters indicate a significant difference (p < 0.05, Tukey’s test).

Figure 2

E3 ligase activity and subcellular localization of Sl1. (A) In vitro expression of MBP-Sl1 and MBP-EV. T: total protein; S: soluble protein; B: before expression; A: after expression. (B) In vitro E3 ligase activity of Sl1 protein. The reaction system included E1, E2, MBP-Sl1, and ubiquitin-His, the replacement of MBP-Sl1 with MBP-EV and the absence of E1, E2, and His-Ub as control. The Western blot was detected with anti-MBP and anti-His. (C) Subcellular localization of GFP-Sl1 and GFP-EV. The GFP-Sl1 was transiently expressed in Nicotiana benthamiana (tobacco with nucleus-located mCherry). Images were pictured by confocal microscope after 48 h infiltration. Bar = 25 μm.

Figure 3

Sl1 positively regulates tomato Cd tolerance. (A) The phenotype of Sl1 mutant lines (sl1-1/2), wild-type (WT), and Sl1 overexpressing lines (Sl1-OE-1/2) under control and Cd stress after 15 d treatment. Bar = 10 cm. (B) The content of hydrogen peroxide (H₂O₂) in the roots of Sl1 mutant lines (sl1-1/2), WT, and Sl1 overexpressing lines (Sl1-OE-1/2) under control and Cd stress after 3 d treatment. (C,D) the image and level of actual quantum efficiency of PSII photochemistry (Φ_PSII) of sl1-1/2, WT, and Sl1-OE-1/2 plants with and without Cd treatment for 15 d. Bar = 1 cm. The data presented here are the average of three biological replicates (±SD). Different letters indicate a significant difference (p < 0.05, Tukey’s test).

Figure 4

Sl1 promotes antioxidant enzyme activities in tomato plants under Cd stress. The activities of SOD, CAT, APX, and GR in the roots of Sl1 mutant lines (sl1-1/2), wild-type (WT), and Sl1 overexpressing lines (Sl1-OE-1/2) under Cd stress for 3 d. The data presented here are the average of three biological replicates (±SD). Different letters indicate a significant difference (p < 0.05, Tukey’s test).

Figure 5

Sl1 decreases Cd content in tomato plants under Cd stress. The Cd content in the shoot (A) and root (B) of Sl1 mutant lines (sl1-1/2), wild-type (WT), and Sl1 overexpressing lines (Sl1-OE-1/2) under Cd stress for 10 d. (C) Cd accumulation in tomato root tips stained by the Cd-specific probe Leadmium^TM Green AM. Bar = 25 μm. (D) Relative fluorescence intensity of Cd staining over tomato root tips of sl1 mutants, wild-type, and Sl1 overexpressing lines after 10 d Cd treatment. The relative fluorescence intensity is normalized to the intensity of wild-type in (C). The data presented here are the average of three biological replicates (±SD). Different letters indicate a significant difference (p < 0.05, Tukey’s test).

Figure 6

Sl1 negatively regulates the transcripts of genes related to heavy metal transportation. The relative expression of CAX3, HMA-A, HMA-B, and IRT1 in the roots of Sl1 mutant lines (sl1-1/2), wild-type (WT), and Sl1 overexpressing lines (Sl1-OE-1/2) under Cd stress for 3 d. The data presented here are the average of three biological replicates (±SD). Different letters indicate a significant difference (p < 0.05, Tukey’s test).