Transcriptome Analysis Reveals the Stress Tolerance to and Accumulation Mechanisms of Cadmium in Paspalum vaginatum Swartz

Abstract

Cadmium (Cd) is a non-essential heavy metal and high concentrations in plants causes toxicity of their edible parts and acts as a carcinogen to humans and animals. Paspalum vaginatum is widely cultivating as turfgrass due to its higher abiotic stress tolerance ability. However, there is no clear evidence to elucidate the mechanism for heavy metal tolerance, including Cd. In this study, an RNA sequencing technique was employed to investigate the key genes associated with Cd stress tolerance and accumulation in P. vaginatum. The results revealed that antioxidant enzyme activities catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and glutathione S-transferase GST) were significantly higher at 24 h than in other treatments. A total of 6820 (4457/2363, up-/down-regulated), 14,038 (9894/4144, up-/down-regulated) and 17,327 (7956/9371, up-/down-regulated) differentially expressed genes (DEGs) between the Cd1 vs. Cd0, Cd4 vs. Cd0, and Cd24 vs. Cd0, respectively, were identified. The GO analysis and the KEGG pathway enrichment analysis showed that DEGs participated in many significant pathways in response to Cd stress. The response to abiotic stimulus, the metal transport mechanism, glutathione metabolism, and the consistency of transcription factor activity were among the most enriched pathways. The validation of gene expression by qRT-PCR results showed that heavy metal transporters and signaling response genes were significantly enriched with increasing sampling intervals, presenting consistency to the transcriptome data. Furthermore, over-expression of PvSnRK2.7 can positively regulate Cd-tolerance in Arabidopsis. In conclusion, our results provided a novel molecular mechanism of the Cd stress tolerance of P. vaginatum and will lay the foundation for target breeding of Cd tolerance in turfgrass.

Keywords: Cadmium; Paspalum vaginatum; PvSnRK2.7; RNA-seq; qRT-PCR.

Figure 1

Enzymatic and non-enzymatic antioxidant activities in leaves of Paspalum vaginatum under stress from Cd. (A) Catalase (CAT); (B) peroxidase (POD); (C) superoxide dismutase (SOD); (D) glutathione S-transferase (GST). Different letters in the column indicate significant differences at p < 0.05, according to Tukey’s test; Vertical bars indicate standard errors of each mean value (n = 3).

Figure 2

Analysis of gene expression by cadmium (Cd) stress tolerance of Paspalum vaginatum. (A) Compiled data of differentially expressed genes (DEGs) at different sampling times in Paspalum vaginatum under cd stress; (B) Venn diagrams of up-regulated DEGs for Cd1 vs. Cd0, Cd4 vs. Cd0 and Cd24 vs. Cd0. (C) Venn diagrams of down regulated DEGs for cd1 vs. Cd0, Cd4 vs. Cd0, and Cd24 vs. Cd0.

Figure 3

Gene ontology (GO) enrichment and KEGG enrichment analysis of DEGs. (A) Go terms of down-regulated DEGs in Paspalum vaginatum under Cd stress. (B) Graphs present 17 KEGG pathways with the highest transcriptional variations out of the down-regulated DEGs in Paspalum vaginatum. (C) Go terms of up-regulated DEGs in Paspalum vaginatum under Cd stress. (D) Graphs present 14 KEGG pathways with the highest transcriptional variations, out of the up-regulated DEGs in Paspalum vaginatum.

Figure 4

Heat map of DEGs involved in glutathione metabolism, metal transporter, and transcription factors. (A) Heat map of DEGs involved in glutathione metabolism; (B) Heat map of DEGs involved in metal transport; (C) Heat map of DEGs involved in transcription factors.

Figure 5

Validation of the expression pattern of 4 selected DEGs in the RNA-Seq by qRT-PCR in Paspalum vaginatum root after Cd treatments. (A) CAX2. (B) NRAMP2. (C) HMA5. (D) SnKR2.7. The lowercase letters above columns represent significant differences between over-expressed and control lines under different treatments (p < 0.05).

Figure 6

Cd resistant phenotypes of the PvSnRK2.7 over-expression lines (Line-1 and Line-2). WT and PvSnRK2.7-OE seedlings were grown on 1/2 MS nutrient solution containing 0 or 80 μM CdCl₂ for 14 d. (A) PvSnRK2.7 gene primers were designed for PCR and RT-PCR identification, then phenotypes were photographed (B), the root length and fresh weight were measured (C). The mean value and standard error were obtained from 3 biological replicates, and the significance difference level p ≤ 0.05 (*), p ≤ 0.01 (**).

Figure 7

Oxidative stress in PvSnRK2.7-overexpressing and control Arabidopsis. (A) Superoxide, (B) hydrogen peroxide, (C) malondialdehyde and (D) glutathione S-transferases contents in PvSnRK2.7-expressed and control plants. Three-week-old seedlings grown on 1/2 MS media with and without 80 μM CdCl₂ were used for experiments. Data indicate the mean ± SE of three independent biological experiments. The lowercase letters above columns represent significant differences between over-expressed and control lines under different treatments (p < 0.05).