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Liang Chen
University of the Chinese Academy of Sciences
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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
Tall fescue, Cd stress, Nitric oxide, RNA-Seq, Metabolite profiling, Detoxification mechanism
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This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfiles.pdf
Table S1. Relative fluorescence intensity in tall fescue roots. The fluorescence was quantified with ImageJ program and registered in fifteen squares of 1000 μm2 each photo. Values are presented as a ration to the untreated Con (n=4). Table S2. Significance at Cd level in Table 1 analyzed by LSD test. The significant difference was presented as capital letters at P < 0.01and small letters at P < 0.05, respectively. Table S3. Significance at NO level in Table 1 analyzed by LSD test. The significant difference was presented as capital letters at P < 0.01and small letters at P < 0.05, respectively. Table S4. Summary of sequence assembly after illumina sequencing. Table S5. Length distribution of the transcripts and unigenes clustered from the de novo assembly. Table S6. Different metabolite levels inT1 treatment and Cd treatment in tall fescue. Table S7. The fold changes of selected metabolitesin T1 vs Cd comparison. Table S8. The level of related DEGs and different metabolites response to T1 treatment vs Cd treatment. Fig. S1. Volcano Plots and Venn diagrams of significantly differentially expressedtranscripts in the tall fescue roots with or without NO treatment under cadmium stress. (a) Volcanoplot in T1vsCd. (b) volcano plot in T2vsCd. (c) Venn diagram analysis in different treatment. (d) Venn diagram analysis between T1vsCd and T2vsCd. Numbers indicate the number of transcripts with significant changes inexpression under different conditions. Overlaps indicate the number of common transcripts differentially expressed, and numbers outside overlaps indicate the number of cultivar or subgroup specific transcripts differentially expressed. There were three regime, comprising Cd, T1, and T2. They respectively presented the tall fescue seedlings were cultivated in 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). Each value is the mean of three replicates. Fig. S2. Histogram of the gene ontology classification analysis of the DEGs in response to theT1 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. S3. The top 10 metabolites according to the VIP values in tall fescue under T1 treatment. 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. S4. Principal component analysis (PCA) of the metabolite profiles in tall fescue roots. The analysis was performed on all the metabolites detected in tall fescue roots under different conditions. 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. S5. The hierarchical cluster analysis (HCA) of the differentially expressed metabolites selected from the integrated analysis between the T1 treatment and Cd 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. S6. The distribution of metabolites in different KEGG pathways. (A) The metabolites in Cd vs Con. (B) the metabolites in T1 vs Cd. There were three regime, comprising Cd, T1 and T2. They respectively presented the tall fescue seedlings were cultivated in 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+ and 200 μM NG-nitro-L-Arg-methyl ester (L-NAME) and 100 μM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (T2 treatment). Fig. S7. The activities of SOD (superoxide dismutase) and APX (Ascorbate peroxidase) in tall fescue roots. Values were given as means ± SD (n = 4). 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. S8. Correlations of expression level analyzed by RNA-Seq platform (y axis) with data resulted from qRT-PCR (x axis).
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See the published version of this preprint at BMC Genomics.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Liang Chen
University of the Chinese Academy of Sciences
Corresponding Author
Badges
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This manuscript passed our Prescreen assessment
Complete author information
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Potential risks to human health
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CURRENT STATUS: Published
View the published article at BMC Genomics.
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Posted 20 Aug, 2020
No community comments so far
Editorial decision: Accept
On 19 Aug, 2020
Editor assigned
On 18 Aug, 2020
Submission checks complete
On 17 Aug, 2020
Editor invited
On 17 Aug, 2020
No comments provided
Editorial decision: Minor revision
On 20 Jul, 2020
Editor assigned
On 07 Jul, 2020
Submission checks complete
On 06 Jul, 2020
Editor invited
On 06 Jul, 2020
No comments provided
Editorial decision: Major revision
On 04 Jun, 2020
Review #2 received
Received 24 May, 2020
Reviewer #2 agreed
On 06 May, 2020
Review #1 received
Received 13 Feb, 2020
Reviewer #1 agreed
On 11 Feb, 2020
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Subject Areas
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License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at BMC Genomics.
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfiles.pdf
Table S1. Relative fluorescence intensity in tall fescue roots. The fluorescence was quantified with ImageJ program and registered in fifteen squares of 1000 μm2 each photo. Values are presented as a ration to the untreated Con (n=4). Table S2. Significance at Cd level in Table 1 analyzed by LSD test. The significant difference was presented as capital letters at P < 0.01and small letters at P < 0.05, respectively. Table S3. Significance at NO level in Table 1 analyzed by LSD test. The significant difference was presented as capital letters at P < 0.01and small letters at P < 0.05, respectively. Table S4. Summary of sequence assembly after illumina sequencing. Table S5. Length distribution of the transcripts and unigenes clustered from the de novo assembly. Table S6. Different metabolite levels inT1 treatment and Cd treatment in tall fescue. Table S7. The fold changes of selected metabolitesin T1 vs Cd comparison. Table S8. The level of related DEGs and different metabolites response to T1 treatment vs Cd treatment. Fig. S1. Volcano Plots and Venn diagrams of significantly differentially expressedtranscripts in the tall fescue roots with or without NO treatment under cadmium stress. (a) Volcanoplot in T1vsCd. (b) volcano plot in T2vsCd. (c) Venn diagram analysis in different treatment. (d) Venn diagram analysis between T1vsCd and T2vsCd. Numbers indicate the number of transcripts with significant changes inexpression under different conditions. Overlaps indicate the number of common transcripts differentially expressed, and numbers outside overlaps indicate the number of cultivar or subgroup specific transcripts differentially expressed. There were three regime, comprising Cd, T1, and T2. They respectively presented the tall fescue seedlings were cultivated in 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). Each value is the mean of three replicates. Fig. S2. Histogram of the gene ontology classification analysis of the DEGs in response to theT1 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. S3. The top 10 metabolites according to the VIP values in tall fescue under T1 treatment. 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. S4. Principal component analysis (PCA) of the metabolite profiles in tall fescue roots. The analysis was performed on all the metabolites detected in tall fescue roots under different conditions. 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. S5. The hierarchical cluster analysis (HCA) of the differentially expressed metabolites selected from the integrated analysis between the T1 treatment and Cd 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. S6. The distribution of metabolites in different KEGG pathways. (A) The metabolites in Cd vs Con. (B) the metabolites in T1 vs Cd. There were three regime, comprising Cd, T1 and T2. They respectively presented the tall fescue seedlings were cultivated in 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+ and 200 μM NG-nitro-L-Arg-methyl ester (L-NAME) and 100 μM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (T2 treatment). Fig. S7. The activities of SOD (superoxide dismutase) and APX (Ascorbate peroxidase) in tall fescue roots. Values were given as means ± SD (n = 4). 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. S8. Correlations of expression level analyzed by RNA-Seq platform (y axis) with data resulted from qRT-PCR (x axis).
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