Comparative transcriptome combined with metabolome analyses revealed key factors involved in nitric oxide (NO)-regulated cadmium stress adaptation in tall fescue

Abstract

Background: It has been reported that nitric oxide (NO) could ameliorate cadmium (Cd) toxicity in tall fescue; however, the underlying mechanisms of NO mediated Cd detoxification are largely unknown. In this study, we investigated the possible molecular mechanisms of Cd detoxification process by comparative transcriptomic and metabolomic approaches.

Results: The application of Sodium nitroprusside (SNP) as NO donor decreased the Cd content of tall fescue by 11% under Cd stress (T1 treatment), but the Cd content was increased by 24% when treated with Carboxy-PTIO (c-PTIO) together with Nitro-L-arginine methyl ester (L-NAME) (T2 treatment). RNA-seq analysis revealed that 904 (414 up- and 490 down-regulated) and 118 (74 up- and 44 down-regulated) DEGs were identified in the T1 vs Cd (only Cd treatment) and T2 vs Cd comparisons, respectively. Moreover, metabolite profile analysis showed that 99 (65 up- and 34-down- regulated) and 131 (45 up- and 86 down-regulated) metabolites were altered in the T1 vs Cd and T2 vs Cd comparisons, respectively. The integrated analyses of transcriptomic and metabolic data showed that 81 DEGs and 15 differentially expressed metabolites were involved in 20 NO-induced pathways. The dominant pathways were antioxidant activities such as glutathione metabolism, arginine and proline metabolism, secondary metabolites such as flavone and flavonol biosynthesis and phenylpropanoid biosynthesis, ABC transporters, and nitrogen metabolism.

Conclusions: In general, the results revealed that there are three major mechanisms involved in NO-mediated Cd detoxification in tall fescue, including (a) antioxidant capacity enhancement; (b) accumulation of secondary metabolites related to cadmium chelation and sequestration; and (c) regulation of cadmium ion transportation, such as ABC transporter activation. In conclusion, this study provides new insights into the NO-mediated cadmium stress response.

Keywords: Cd stress; Detoxification mechanism; Metabolite profiling; Nitric oxide; RNA-Seq; Tall fescue.

Fig. 1

The NO content in tall fescue roots. Tall fescue roots were loaded with the sensitive fluorescent dye DAF-FM DA. a and b represent bright and fluorescent field, respectively. The lower corner of each photo has a scale bar in 50 μm length

Fig. 2

The Cd content in roots of tall fescue under four different treatments. There were four regimes, comprising Control (Con), Cd, T1 and T2 treatment. Values are given as mean ± SE (n = 3). Data about Cd, T1 and T2 treatment were analyzed using one-way Analysis of Variance, followed by LSD test. Asterisks (*) indicate the significant difference at P < 0.05

Fig. 3

Scatter plot analysis of the DEGs in response to the T1 treatment in tall fescue roots. The tall fescue seedlings were cultivated in 1/2 Hoagland solution with 50 mg/L Cd²⁺ (CdCl₂•2.5H₂O) (Cd treatment) and 1/2 Hoagland solution with 50 mg/L Cd²⁺ and 200 μM SNP (T1 treatment), respectively. Each value is the mean of three replicates

Fig. 4

The hierarchical cluster analysis (HCA) of the metabolite profiles in tall fescue. There were four treatment regimes in this study, including the Con, Cd treatment, T1 treatment, and T2 treatment, and each regime had three replicates. They respectively presented the tall fescue seedlings were cultivated in 1/2 Hoagland solution (Con), 1/2 Hoagland solution with 50 mg/L Cd²⁺ (CdCl₂•2.5H₂O) (Cd treatment), 1/2 Hoagland solution with 50 mg/L Cd²⁺ and 200 μM SNP (T1 treatment), and 1/2 Hoagland solution with 50 mg/L Cd²⁺, 200 μM L-NAME and 100 μM c-PTIO (T2 treatment)

Fig. 5

Histogram of the differentially expressed related genes and metabolites in response to the T1 treatment vs Cd treatment in tall fescue. The tall fescue seedlings were cultivated in 1/2 Hoagland solution with 50 mg/L Cd²⁺ (CdCl₂•2.5H₂O) (Cd treatment) and 1/2 Hoagland solution with 50 mg/L Cd²⁺ and 200 μM SNP (T1 treatment), respectively. Each value is the mean of three replicates

Fig. 6

The simplified schematic of the related region of some pathways in the T1 treatment vs Cd treatment in tall fescue. The tall fescue seedlings were cultivated in 1/2 Hoagland solution with 50 mg/L Cd²⁺ (CdCl₂•2.5H₂O) (Cd treatment) and 1/2 Hoagland solution with 50 mg/L Cd²⁺ and 200 μM SNP (T1 treatment), respectively. The green labels show the down-regulated genes or metabolites, and the red labels show the up-regulated genes or metabolites. The solid arrows indicate that the biosynthesis of the metabolite indicated is a single approach. The dotted arrows indicate the biosynthesis of the metabolite indicated is a multipath

Fig. 7

A proposed model of nitric oxide (NO)-regulated cadmium stress response in tall fescue