Metabolome and transcriptome association analysis revealed key factors involved in melatonin mediated cadmium-stress tolerance in cotton

Abstract

Cadmium (Cd), a non-essential element for plant, is a ubiquitous and highly toxic heavy metal, seriously endangering agricultural production and human health. As a nonedible economic crop, cotton (Gossypium hirsutum L.) has great potential in remediation of Cd contaminated soil, but its underlying mechanism is still unknown. Melatonin (MT), as a plant growth regulator, is involved in alleviating Cd toxicity in some plants, but the molecular mechanisms of MT-mediated Cd detoxification in cotton are largely unknown. This study investigated the possible molecular mechanisms of the MT-mediated Cd detoxification in cotton seedlings by comparative transcriptomic and metabolomic analyses. The results showed that the cotton seedlings were dwarfed and the leaves were wilted and yellow under Cd stress. The application of 50 µmol L^-1 MT significantly increased the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content under Cd stress, but 100 µmol L^-1 MT significantly decreased SOD activity, while increased ascorbate peroxidase (APX) activity significantly. The addition of 100 μmol L^-1 MT significantly increased Cd concentration in the shoots and roots under Cd stress. RNA-seq analysis showed that 5573, 7105, 7253, 25, 198, 9 up-regulated and 6644, 7192, 7404, 9, 59, 0 down-regulated differentially expressed genes (DEGs) were identified in the comparisons of CK vs T1, CK vs T2, CK vs T3, T1 vs T2, T1 vs T3 and T2 vs T3, respectively. It was revealed that MT promoted the expression of certain related genes under Cd stress, and the effect of 100 µmol L^-1 MT was better. Moreover, UPLC-MS/MS widely targeted metabolites analyses showed that 195, 150, 150, 12, 24, 59 up-regulated and 16, 11, 23, 38, 127, 66 down-regulated differentially accumulated metabolites (DAMs) were changed in the CK vs T1, CK vs T2, CK vs T3, T1 vs T2, T1 vs T3 and T2 vs T3, respectively. It was revealed that MT induced the synthesis of alkaloids and flavonoids, and inhibited or reduced the synthesis of lipids, amino acids and their derivatives. The comprehensive analyses of transcriptomic and metabolic data showed that 33 DEGs and 4 DAMs, 46 DEGs and 16 DAMs, and 1 DEGs and 1 DAMs were dominantly involved in the pathways of valine, leucine and isoleucine degradation, ABC transporter, alpha-linolenic acid metabolism, respectively. It was revealed that there were three major mechanisms involved in MT-mediated Cd detoxification in cotton, including the enhancement of antioxidant capacity regulated by APX, flavonoids and alkaloids; accumulation of secondary metabolites related to Cd chelation, such as amino acids and derivatives; and regulation of cadmium ion transportation, such as ABC transporter activation. In conclusion, this study provides new insights into the MT-mediated Cd stress response.

Keywords: 2-hydroxymelatonin; Cd stress; Detoxification mechanism; Gossypium hirsutum; RNA-Seq; UPLC-MS/MS.

Figure 1

The number distribution, venn diagram and KEGG pathway enrichment map of DEGs in different sample group. (A), The number distribution of DEGs among CK vs T1, CK vs T2, CK vs T3, T1 vs T2, T1 vs T3, T2 vs T3; (B), Venn diagram of DEGs among CK vs T1, CK vs T2, CK vs T3, T1 vs T2, T1 vs T3, T2 vs T3; (C), KEGG pathway enrichment map of DEGs among CK vs T1, CK vs T2, CK vs T3. CK refers to the control, T1 refers to 200 µmol L⁻¹ Cd treatment, T2 refers to the treatment of 50 µmol L⁻¹ MT+200 µmol L⁻¹ Cd, T3 refers to the treatment of 100 µmol L⁻¹ MT+200 µmol L⁻¹ Cd.

Figure 2

The number distribution, venn diagram and KEGG pathway enrichment map of DAMs in different sample group. (A), The number distribution of DAMs among CK vs T1, CK vs T2, CK vs T3, T1 vs T2, T1 vs T3, T2 vs T3; (B), Venn diagram of DAMs among CK vs T1, CK vs T2, CK vs T3, T1 vs T2, T1 vs T3, T2 vs T3; (C), KEGG pathway enrichment map of DAMs among CK vs T1, CK vs T2, CK vs T3, T1 vs T2, T1 vs T3, T2 vs T3. CK refers to the control, T1 refers to 200 µmol L⁻¹ Cd treatment, T2 refers to the treatment of 50 µmol L⁻¹ MT+200 µmol L⁻¹ Cd, T3 refers to the treatment of 100 µmol L⁻¹ MT+200 µmol L⁻¹ Cd.

Figure 3

Correlation quadrant diagrams representing association of DEGs and DAMs. (A), CK vs T1; (B), CK vs T2; (C), CK vs T3; (D), T1 vs T2; (E), T1 vs T3; (F), T2 vs T3.

Figure 4

KEGG enrichment analysis pvalue histogram of DEGs and DAMs. (A), CK vs T1; (B), CK vs T2; (C), CK vs T3; (D), T1 vs T3. The abscissa represents metabolic pathways, and the ordinate represents the enriched pvalue of DEGs (red) and DAMs (green), which is represented by -log (Pvalue). The higher the ordinate, the stronger the enrichment degree.

Figure 5

The network analysis of DEGs and DAMs in response to Cd stress and the addition of MT in cotton seedling leaves. (A) the correlation network of DEGs and DAMs involved in the pathway of valine, leucine and isoleucine degradation; (B) the correlation network of DEGs and DAMs involved in the pathway of ABC transporter; (C), the correlation network of DEGs and DAMs involved in the pathway of alpha-linolenic acid metabolism; ●The red circle represents DEGs; ♦the green square represents DAMs, the solid red line represents positive correlation, and the dashed black line represents negative correlation.

Figure 6

The DEGs and DAMs involved in the pathway of valine, leucine and isoleucine degradation in response to Cd stress and the addition of MT. BCAT, branched-chain amino acid aminotransferase; BCKDHA, 2-oxoisovalerate dehydrogenase E1 component alpha subunit; DLD, dihydrolipoamide dehydrogenase; DBT, 2-oxoisovalerate dehydrogenase E2 component (dihydrolipoyl transacylase); IVD, isovaleryl-CoA dehydrogenase; ECHA, enoyl-CoA hydratase; MCCC, 3-methylcrotonyl-CoA carboxylase alpha subunit; ACAT, acetyl-CoA C-acetyltransferase; HMGCS, hydroxymethylglutaryl-CoA synthase; HIBCH, 3-hydroxyisobutyryl-CoA hydrolase; HIBADH, 3-hydroxyisobutyrate dehydrogenase; AGXT2, alanine-glyoxylate transaminase/(R)-3-amino-2- methylpropionate-pyruvate transaminase; ALDH, aldehyde dehydrogenase (NAD+); ALDH6A1, malonate-semialdehyde dehydrogenase (acetylating)/methylmalonate-semialdehyde dehydrogenase; ACAA, acetyl-CoA acyltransferase. The blue pattern represents the DEGs or DEGs that changed under Cd stress and the addition of MT. The rectangle is divided into three equal parts (the left of rectangle represents DEGs or DEMs in CK vs T1, the middle of rectangle represents DEGs or DEMs in CK vs T2, the right of rectangle represents DEGs or DEMs in CK vs T3). The color in the rectangle represents the DEGs or DEMs are regulated under Cd stress and the addition of MT (red indicates up-regulation, green indicates down-regulation, blue indicates both up-regulation and down-regulation, white indicates neither up-regulation nor down-regulation).