Sucrose synthase gene SUS3 could enhance cold tolerance in tomato

Abstract

Tomatoes are susceptible to damage from cold temperatures in all stages of growth. Therefore, it is important to identify genetic resources and genes that can enhance tomato's ability to tolerate cold. In this study, a population of 223 tomato accessions was used to identify the sensitivity or tolerance of plants to cold stress. Transcriptome analysis of these accessions revealed that SUS3, a member of the sucrose synthase gene family, was induced by cold stress. We further investigated the role of SUS3 in cold stress by overexpression (OE) and RNA interference (RNAi). Compared with the wild type, SUS3-OE lines accumulated less MDA and electrolyte leakage and more proline and soluble sugar, maintained higher activities of SOD and CAT, reduced superoxide radicals, and suffered less membrane damage under cold. Thus, our findings indicate that SUS3 plays a crucial role in the response to cold stress. This study indicates that SUS3 may serve as a direct target for genetic engineering and improvement projects, which aim to augment the cold tolerance of tomato crops.

Keywords: SUS3; cold stress; sucrose synthase; tomato; transcriptome.

Figure 1

Differentially expressed genes (DEGs) in cold-sensitive and cold-tolerant tomato varieties. (A) Partial cold-sensitive and cold-insensitive tomato varieties. (B) Volcano map of DEGs. (C, D) Significantly enriched GO terms (C) and KEGG pathways (D) of DEGs. (E) Heatmap of gene expression related to sugar metabolism. (F) Gene expression related to sugar metabolism by qRT-PCR.

Figure 2

Molecular characterization of SUS3. (A) Subcellular localization of the SUS3 protein in protoplasts. (B) Agrobacterium strains carrying the GFP-SUS3 were digested into protoplasts in tobacco leaves. Bright-field, green fluorescent, red fluorescent, and merged images are shown.

Figure 3

Relative expression pattern of SUS3 under abiotic stress and hormone induction. (A–E) Expression pattern of SUS3 in ABA, ethylene, drought, cold, and salt. Four-week-old seedlings were stressed with ABA (100 μM), ethylene (200 μM), drought (dehydration), cold (4°C), and salt (400 mM of NaCl) treatments for 0, 1, 3, 6, 12, and 24 h, respectively. The relative expression levels were determined using qRT-PCR. The expression levels of the appropriate controls were set to a value of 1. Data are presented as mean ± standard deviation of three biological replicates. Asterisks indicate statistically significant differences between transgenic lines and wild type. *, p < 0.05; **, p < 0.01.

Figure 4

Overexpression of SUS3 improves the cold tolerance in tomato. (A, B) Phenotype of transgenic seedlings expressing abnormal levels of SUS3 and WT seedlings under cold stress conditions. The SUS3-OE lines (OE1-2, OE2-4, and OE3-2), SUS3-RNAi lines (Ri1-1, Ri8-3, and R7-2), and WT seedlings were grown at 4°C for 7 days. (C) Relative expression levels of SUS3 in transgenic lines and WT. Comparison of MDA content (D), soluble sugar (E), relative electrolyte leakage (F), and proline (G) in SUS3-OE lines and WT under cold stress and optimal conditions. Data are presented as mean ± standard deviation of three biological replicates. Asterisks indicate statistically significant differences between transgenic lines and wild type. *, p < 0.05; **, p < 0.01.

Figure 5

Superoxide radicals(O₂ ^−.) accumulation and related enzyme activity of SUS3-OE lines and WT under cold stress and optimal conditions. (A) NBT staining used to detect the accumulation of superoxide radicals. The SUS3-OE lines (OE1-2, OE2-4, and OE3-2) and WT under cold stress and optimal conditions were incubated at 25°C in the dark for 2 h. Comparison of SOD content (B), APX (C), CAT (D), and POD (E) in SUS3-OE lines and WT grown under cold stress and optimal conditions. Data are presented as mean ± standard deviation of three biological replicates. Asterisks indicate statistically significant differences between transgenic lines and wild type. *, p < 0.05; **, p < 0.01.